CA2203265A1 - Production and use of human and plant methyltransferases - Google Patents

Abstract

An isolated recombinant human L-isoaspartyl/D-aspartyl protein methyltransferase is obtained by overexpression of cDNA coding for isozyme II
in an E. coli strain, and a cDNA clone of the wheat enzyme and a purified enzyme from wheat are obtained. These enzymes are useful in treatment of medical conditions and diagnosis of disease associated with an increase in L-isoaspartyl/D-aspartyl residues of polypeptides in a tissue.

Description

CA 0220326~ 1997-04-21 PRO~U~IION AND USE OF
HUMAN AND PLANT METHYLTRANSFERASES
BACKGROUND
Field of the Invention The present invention relates to the r ~u~i and use of methyltransferases. More ~,u~cifi 11y, the invention relates to the, uduLIiu~l, r.,.iriLai and use of ,. ' : human L-i~oaa~ yllD-aspartyl protein hyllldlla~ ase~ ân isolated p~'~,,uLl.,~ coding for a plant L iaoaa~Jdl l~l protein methyltransferasa and a purified plant L-iaGaa,ua, L~l protein lll~ Ih~lllal,arL,~se.
BaGIu,luu,,d of the Invention - 10 Proteins underg~ " ~ l~nc~u~ Lh~, d~ driven changes over time that can result in lle~ aatd functionality (Harding, J.J. (1985) Adv.Protein Chem. 37:247-334). A common form of such damage is the r y"~dli~, dL ' :~ c,lion, and, liu.. of aa~ rl and aspartyl residues (Clarke, et al. (1992) "Stability of Protein rhallllac~ .ls, Part A: Chemical ând Physical Palh. ~ù of Protein D6UI ' " pp1-29, Plenum Press, New York). In most cells, proteins co..i ~ L j5GaSUaIlYI and D-aspartyl residues are r~co~, ldby theprotein-L-ia~aa,ua,l~l~(D-aspartate)O-,,,~ll,;l~,a.,a~,aae(E.C. 2.1.1.77) whichcatalyzestheirmethyleaL~,i6LaLio, 1 'add~", et al. (1982) Proc.lU~tLAcad.ScL USA 79:2460-2464, Murray, et al. (1984) JR;olrhPm 259:10722 10732, and Aswad (1984) J~;qlChPm 259:10714-10721). The enzymic III~Lh~laLiuil reaction " . ~CIL,~La the first step of a process that results in the Cbllv~,. ~ of these altered aspartyl residues to normal L-aspartyl residues (~' 'add~", et al. (1987) Proc.lUatl. ~cP~lsri USA 84:2595-2599, Johnson, et al. (1987) J R;^lClr ~ 562~-5629, Lowenson, et al. (1991) J.~;~/Chem. 266:19396-19406, Lowenson, et al. (1992) JR;7lchpm 267:5985-5995).
This reaction prevents the ac.u,,,ulalian of proteins CGnl ~ ;aulll~H~d and (ac~",i~d aspartyl residues and may be an ;IIIP(~IL~IIL C-~ JI~- --I in limiting the d~ lllal effects of the aging process. In bacteria, it has been shown that the enzyme is crucial to al~liur,aly phase and heat shock survival since deletion ",uld6ùns decrease survival by up to 100-fold (Li, et al. (1992) Proc.lUatl.Acad.Sci. USA 89:9885-9889) L-;aGda~dl I ;llD-aspartyH"~Illylll dna~el aa~a have been ~ lla6~1y studied in "~ 1" ~ tissues. Two similar activities have been isolated from bovine brain (Aswad, et ab (1983) J. Aleurochem. 40:1718-1728) and human elyi' uCyl~a (Gilbert, et al. (1988) Bi~ lrv 27:5227 52331 Ota, et al. (1988) Biochem. Biophys. ~es. Commun.
151:1136-1143). These isozymes are IllGnblll~, poly~Je~-6des of about 25,000 Da and have similar catalytic ~J~u,uelli~:S, but differ by about 1 pH unit in ;aoel~LL,i~ point (Ota, et al. (1988) Eiochem. Bioph)~s. Res. Commun.
151:1136-1143). A third activity has been noted, with an ;aoeleLl,iL point between isozymes I and ll, but the cGr,~apc ' ~ isozyme has not been isolated or chdlaGL~ d (Ingrosso, et al. (1991) Adv. Exper. Med. Biol. 307:263 276). The complete amino acid seq.~e"ces of isozyme I from bovine brain (Henzel, et al. (1989) J. Biol. Chem.
264:15905-15911) and human erythrocytes (Ingrosso, et al. (1989) J. ~iol. Chem. 264:20131-21039) as well as most of that of isozyme ll from human elyll"ucytes (Ingrosso, et al. (1991) Biochem. Bioph)/s. Res. Commun.
175:351-358) have been d~l~,ll .~~ The sequence of the human isozyme I is 96% identical to the bovine enzyme.
The amino acid seuu - .es of the two human isozymes appear to be identical with the exception of two amino acids CA 0220326~ 1997-04-21 W O96/12797 PCT/US9~tl3691

-2-at the C-terminus which may account for the d;ll~ ce in ;~G~I~L~ point (Ingrosso, et al. (1991) Biochem. Biophys.
~es. Commun. 175:351-358).
Southern blot analysis suggests that both isozymes are products of a sin~le ~ene (Ingrosso, et al. (1991) ~iochem. ~ioph~s Res Commun. 175:351-358). ~ 9 encoding enzymes similar to isozyme I in rat brain and mouse testis have recently been determined (Sato, et al. (1989) Biochem. Biophvs. Res. Commun. 161:342-347, Romanik, et al. (1992) 6ene, 118:217-222).
Two cDNA clones cu". r " " to the mRNAs for two isozymes of the human Li~,oas~.d,lyllD-aspartyl protein carboxyl I ' hylL~ an .Ibrase (EC 2.1.1.77) (MacLaren, et al.(1992) Bio~hem. Biophys. Res. Commun. 185, 277-283) have been ~, cc' The DNA sequence of one of these (pRK1) encodes the amino acid sequence of the C-terminal half of the human ~. yll"~ y~e isozyme 1. The other cDNA clone (pDM2) includes the complete coding reyion of the more acidic isozyme ll. With the exception of potential p l~ . I ' sites at amino acid residues 119 and 205, the deduced amino acid s3~, es differ only at the C-terminus, where the -RWK sequence of isozyme I is replaced by a -RDEL sequence in isozyme ll. The latter sequence is identical to a mammalian endoplasmic reticulum r~tention signal. The missing portion of the coding region for pRK1 is assumed to match that of pDM2. The presence of ~ ",ali~d splicing suggests the existence of a third isozyme having a R C-terminus. Finally, evidence has been ~,.tS~."I~d for the existence of three genomic pGI~ h;~"", in the human gene for this enzyme. Residue 22 can be either isoleucine (I) or leucine (L); residue 119 can either be isoleucine or valine (V); and residue 205 can either be Iysine (K) or arginine (R) (Tsai and Clarke (1994) Biochem. Biophvs. Res. Commun. 203:491-497). Thus, each isozyme described above can exist in at least eight forms (l22lll9K2o5~ 122l11gR20s~ l22V119K205~ l22V119RZ05 L22l11sK20s~ L22l119R205~ L22V119K205~ L22V119R205) However, in the above studies, the DNAs coding for the human ul~hyll~dll~l~làs~ were not efficiently ~A~ sed due to the lack of suitable recombinant ~ systems. Thus, large amounts of hc, ~ OIJ~ enzyme have not been p~G.h~li;,ly isolated. M~ L C~,a~e has been purified from bovine brain where 3.7 mg was obtained in 5 steps from 0.58 kg of cerebral cortex (Aswad, et al. (1983) J.,'l'~ . ~' 40:1718-1726.). The human enzyme has been more difficult to purify, i.e., only about 0.1 mg of protein was obtained in 5 steps from 0.22 liters of blood (Gilbert, et al. (1988) Bi ' ~ 27:5227-5233.). Accu,d ~1y, efficient ,uuliril,dO~m systems to produce large UUd~ s of isoaspà,lyl ",~lh~ lc~dses on an industrial scale have h~.~lulu,~ not been available.
In plants, a protein L-i:,oas,uall~l ",~lh~lll r~ld;~e activity has been identified in both the monocots and the dicots as well as the green algae, Cf ' ~ osl ' Jlii(Mudgett, et al. (1993) Bi~ lv 32:11100-11111). Although some ~"~y",ali.. activity is present in most plant organs, the levels can vary con~id~ldbly.
I"l~,e,li"gly, the highest level of L-isoaspartyl Ill~h~l~ldll~l~ld5e activity is found in seeds (Mudgett, et al. (1993) B~ocr'~ i,y 32:11100 11111). Moreover, the in vitro formation of carboxyM"~ll,yldled proteins in the soluble fraction of seeds in the absence of Plo~ J~ peptide lub~ldl~5 suggests that methyl-accepting ~uL;,ll-dl~;, exist for the ",~O,yll,ansr~,àse in vivo (Trivedi, et al. (1982) Eur. J. Biochem. 128:349 354 and Mudgett, et al. (1993) Bierhe~ r~/ 32:11100 11111).

CA 0220326~ 1997-04-21

3-The level of iaoaa~Jalldl~ in proteins hâs been used as an indication of the level of damage to the proteins.
Methods for determining the iiodaudllyl content of proteins using ;aoaapdllyl methyl lldll;.lblase enzymes are disclosed in U.S. Patent No. 5,273,886 to Aswad, the disclosure of which is hereby incu.rJ~dled by r~f~
BRIEF DESCF~ ON OF THE FIGURES
FIGURE 1 shows cr t- u~.liun of the human L-;.~uaapdl I~llD-aspartyl ~ h~ltl ~uldae expression plasmid, pDM2x of the present invention. A 107bp KpnlJI/arl fragment is removed from plasmid pDM2 and replaced with a synthetic polylinker cr ~ ribosome binding and initiator sites.
FIGURE 2 shows the novel polylinker fragment used in the present invention. The polylinker contains multiple cloning sites (Kpnl, Khol, Xb~l, BamHI, IJhel, llcol) and a strong, Ol~alyulil, ribosomal binding site (Shine, et al. (1974) Proc. Al~tl. Acad. Sci. USA 71:13421346).
FIGURE 3 shows the nu~'u~,lid~ and deduced amino acid sequence of the human L ;aoaapd.lyllD aspartyl ..~ll.yllld,.ar~,dae ~,ull plasmid, pDM2x, of the present invention (SEQ ID NO:8).
FIGURE 4 shows the effect of isopropyl,~D-th g ' rYI ---1 (IPT6) Gonc~lllldi on the level of human L-;~oas,ud,lyllD-aspartyl ",~LI,tlll l~l#a~ ~"udu"li~.rMn E. co/i.
FIGURE 5 shows E. coli growth percent of total soluble protein obtained as human methyltransferase, and yield of human -:I~yll,d"~le,dae in the pDM2x ~A,UI. system of the present invention as a function of time after IPTG ;~.du~,i FIGURE 6 shows ~"lilll;~di- of the I ~ui - sulfate ~J" ~il step according to the present invention.
FIGURE 7 shows L, lillli~dliUn of a sulfate pl~L~;,u;ldliun lldl~liullaliun of human L;aoaa,uallyllD-aspartyl Ill~lLyllldl,areldae from the protamine sulfate clarified 5~ "lldldlll according to the present invention.
FIGURE 8 shows the anion exchange column purification step of the human L;aoda,udllyllDaspartyl hrllld~al~dae according to the present invention.
FIGURE 9 shows the pH d~pr ' for car lldlillg purified human L-;aoaa,udllyllD aspartyl Ill~lhylll~.,.al~ldae according to the present invention.
FIGURE 10 shows el~llusp~dy mass spectral analysis of purified l.~ ' I human ",~lllyllld,~al~,dae of the present invention. (A) A portion of the spectrum of material purified using ~;:' ILI~;Iol. (B) A portion of the spectrum of material purified when 15mM ~ .d,uto~lha~ol was s.,b~l;luled for 0.1 ,uM d;liulh,~;lol in buffer A.
FIGURE 11 shows the .IIIIu~ ' I ' Ld""e spectrum of the purified human L;aoda,ud,lyllD-aspartyl ..,~II,yllld,lal~'dse of the present invention.
FIGURE 12 shows the puDliLdliull of wheat germ L-iaoda,ua,Lyl ll,~lhyll,d"al~ldse according to the present invention. (A) DEAE 52 anion exchange cellulose ll~dllll~ . (B) Reverse ?~ sulfate gradient solubilization ed I. (C) Sephacryl S-200-gel filtration.
FIGURE 13 shows the Dul~ l,.,,,lide analysis of the purification of L-isoaspartyl methylll dllal~' dae from wheat germ according to the present invention.
FIGURE 14 shows the DNA s~ ;"J strategy of the wheat germ u,~ll,yllld"al~,ase cDNA insert employed in the present invention.

CA 0220326~ l997-04-2l FIGURE 15 shows an alignment of the sequenced peptide fragments of L~iaoaalJd~ i methyltransferase from wheat yerm and its predicted amino acid sequence from pMBM1.
SUMMARY OF THE INVENTION
One particular objective of the present invention is to provide an isolated - - ' I human L-5 ;aoas~al IyllD-aspartyl protein melh~ltl ' aae by expression of the cDNA encoding the enzyme. Surprisingly, this e..~" system permits an ~AIn ~ efficient large scale, ~ of a pure recombinant enzyme. The structure of the recombinant enzyme is different from that of the purified enzyme from human c.rlh,l,..1l~;, only at the N-terminal alanine residue where the recombinant enzyme is not modlfied by the post-translational modification of ac~Lyldliùm Thus, this enzyme is suitable for use in human studies without the potential problem of ~ y.
Other ~b, ~ s of the present invention include the i'~."ti~ n of cDNA encoding wheat L-i~oa~.d,l),l protein l~lh~ ,ase, and the provision of a purified plant L -, Ijl protein ",~lh jlt, f~ laae from wheat.
High levels of methyltransferase are found in wheat, especially in seeds.
Still other .bJ _~ U; of the present invention are to provide a 1' . ~ , dliU~ containing the enzyme to treat disorders resulting from protein d~,addliun, and to provide an analytical tool for quality control of protein and peptide ~JI,a""ac~.tiL ' and for diagnosis of disease states 7 ohd with protein degradation. Other Db; Li.~.;, of the present invention will become apparent to one having ordinary skill in the art upon reference to the ensuing detailed description of the invention.
Namely, one aspect of the present invention is an isolated "~l ' l human L-;aoaa,.a,lyllD-asparty protein Ill~lhyllrdlls~ldae obtained by e.~,.l of a p~ llucl~.vLidd having a sequence selected from the group ce ~;,li- 9 of SEQ ID NO:1 (isozyme 1) and SEQ ID NO:2 (isozyme ll), each including a total of 17 nucleotide , coding foreight amino acid ~e, ~s of isozyme I or ll, i-e-, 122l119K205~ 122l119R205~ l22V119K205 22V119R205~ L22l11sKZOs~ L22l119R205~ L22V11gK20s~ and L22V11gR205 as described earlier.
Another aspect of the present invention is an isolated ,. r ' l human L-;soas~.d,l;llD-aspartyl protein llldlla~l..dse having an amino acid sequence selected from the group c : E of SEQ NO:3 (isozyme 1) and SEQID NO:4 (isozyme ll), each including eight amino acid ~eu,uPI~ces as above.
Another aspect of the present invention is an isolated pol~, ' lidd having the coding sequence of the sequence indicated as SEQID NO:5, which codes for a plant L-;aoaspa,l;l protein l"~lI,ylLIa"a~,aae. The 690 base pairs of this sequence beginning with the ATG codon at base numbers 117-199 and ending with the AGC codon prior to the TGA codon at positions 807-809 represent the coding sequence of this F'~ e.
Another aspect of the present invention is an isolated ~. ' I plant L-;sûaalJal Lyl protein ",~lhyll,dr,a~dldse obtained by ~ aa;~m of a pol~ ctide having the sequence indicated as SEQID NO:5.
Another aspect of the present invention is a purified plant L-;audapallyl protein Ill~Lll~llrarla~dlaae from wheat germ having the amino acid sequence indicated as SEQID NO:6 or 7.
Another aspect of the present invention is a method of ,.r ' lly producing human L-isoas~Ja,lyllD
aspartyl protein Ill~LllylLldlla~ldse, culll~JGably.

CA 0220326~ 1997-04-21 Cyj~ly a piasmid such as plasmid pDM2 (E~ accession # S374g5~ that contains the full coding re~ion of human L-;~oa~ud, IyllD aspartyl protein :hylt, ~ . aS~, using -'4, ' ~iJes, to provide multiple cloning sites, an efficient ribosome bindiny site, and a strong translational initiator reynion, said initiator region being designed to function in bacterial andlor eukaryotic expression system:
ll . C~ i"g the c ~ d vector into a host that contains an isopropyl ,~ D-thiogalaclu,.~dnoside (IPTG) inducible T7 polymerase gene; and inducing 0.3,La~ - of the :~ .u~,e with IPTG, whereby the m~tll~ltians~c~as~ is produced.
Also, any system producing T7 polymerase gene can express the methyltransferase.This ~.",.~ r method is -~ p, " ' ' to any variants of the h~ Ce.a~e. The enzymeis, ~telabl~ obtainedfrom an ~ .,u~sLdhuman cDNA in E. coli such as BL21(DE31 grown in LB Broth. Further efficient and economical expression is achieved using a richer media, e.g., terrific broth (S ' . k, et al. (1989) Cloning: A l: ' dloly Manual," Cold Spring Harbor LabGrdloly), which results in a higher final cell density and a longer exponential growth and ",~th~lt,d,.;,r~ ,a ,e ~ ud,..,liu.. phase. In the present invention, the final result can be a plepdldliun where the human Ill~lh~ all~r~la~e makes up about 10-30% of the totai bacterial solubl ~ protein.
Another aspect of the present invention is a method of purifying ,~ produced human L-i.oa~,ua.lyllD-aspartyl protein ...~,II,~IIl.u.;,~,ui.e present in a Iysed bacterial extract in which the m~ll,yll, - 'elase ~,~u,, has been r ~ d, s 1, adding a nu..l~ulid," .,~;~.ila"l such as 1~0 sulfate or pol~lh~h,n~;..line to the extract to remove DNA 0 present in the extract s -~ .. .l to removing the cellular debris;
prel,;y;ldlilly the ",~ llldnsi~,dsd with: sulfate;
removing the ammonium sulfate by dialysis; and purifying the I Ih~dllal\~elase from the dialysate using anion-exchange ..h.ulllaluy.aphy under novel cor,.l'~iun~
Based on this novel purification, it is possible to obtain about 50 mg of enzyme from 4 liters of bacterial culture. The success of a single column in purifying this enzyme is attributed to finding corl~ s where the enzyme can be weakly bound to the column and eluted isG~,IdtiL 'l~ in starting buffer without the ar,' liul. of a salt gradient. It is found that a DEAE-cellulose column not fully , ' ' dl~d with a phosphatelEDTA buffer gave the best I- dCIiUllaliUI~.
~0 Another aspect of the present invention is a method of purifying plant L-;soa;"ua, Iyl protein I,ylllàll~ld~e from wheat, cu",,u,i~;.,y.
obtaining a crude cytosol from raw wheat germ;
rld~,liolldlilly the crude cytosol by DEAE-cellulose clllullldluy~dplly~
adding Im sulfate to the pooled active fractions in the presence of â protein carrier;
rlà(,liOIIa; ~ the resulting material by reverse a.. ", " sulfate gradient s-' ' ' liùu1, and CA 0220326~ 1997-04-21 W O 96/12797 PCTAUS9~/13691 purifying the pooled active fractions by gel filtration chromatography, whereby the methyltransferase is purified as a i.. 28,000 Da species.
Another aspect of the present invention is a method of treatment for a medical ~o ~ ass~.~;al~d with an increase in L~ llD-aspartyl residues of P~l~r ~ 1- ' in a tissue, s , i ~, ' i,.~, to the tissue an 5 amount of methyltransferase, with or without S-adenosylmethionine, sufficient to convert said L-i~uas~,a, Iyl;D-aspartyl residues to L-aspartyl residues in the tissue.
According to another aspect of this preferred embodiment, there is provided a method of diagnosis of disease states where L-;~oa;"ua,lyllD-aspartyl residues are accumulated, comprising measuring the content of L-i~Oa~lal I~llD-aspartyl residues accumulated in a disease aS~O-.ial~.d protein, by using Ill~lhJd~lall;,iLla;,e as a probe.
Another aspect of the invention is a method of determination of degradation of pha"~ I F~ es, cr , i:,;"~ ",ea;,u~ ~ the content of L-iaua~,ual l~l and D-aspartyl residues in the ~ H~ Iid~, by using m~lhtlt,a"~,a~e as a probe.
Stili another e L-' : of the invention is a 1' ",ae.,.,li,,dl p~,ualaliun for l,~al",~"l of a medical condition acsnri-lPd with an increase in L-i~a~.a,lyllD aspartyl residues of polypE,.IiJes in a tissue, CGIII~ ;lly 15 human L-;aoaspd,l;llD-aspartyl protein ll~lh11ll. 5 la~",.~ hl~ with its substrate S-adenG~yh"~ ine, and 1 h "~ac~.,li"ally Zc--r ~' carriers.
Since large amounts of the enzyme can be obtained by the present invention, the commercial utility of this enzyme becomes -rr~ ~ ~ The Ill~:lhyl~ra,-;,r~,a;,ecan be used as a sensitive analytical probe of L-i~oaspdllyl and D-aspartyl residues in quality control of protein and peptide ,,~L.~ t;~'l` The uses in medical diagnostics for 20 assaying for altered proteins and peptides in biological fluids such as the B-amYIûid product (Roher, A.E., et al.
(1993) JR;~Ichem 268:3072-3083) is possible. Because the c ~ O human, :b~,ll,dr,sl~,dse is limited to the cytosol (Clarke, S. (1985) Annu Rev Biochem 54:479 506), damaged proteins in the ~,~l, r " ' - e,,v;,u,,,,,~
cannot undergo repair catalyzed by this enzyme. Thus, injectable and topical Ih~,, li,, ~..~Jaldliuns using the ~"~LI,yll,a";,ie,ase and its substrate S ad -sy' thionine are useful. In particular, the u~ .l"ess of this enzyme 25 in repairing damaged proteins in skin such as collagen and elastin whose d~yl~d liun may collLIi' le to the aging of this tissue is advantageous.
DETAILED DESGhl~llON OF THE PF~E~t~E~ EMBODIMENTS
Overexpression of the Human L-lsoaspartl~llD-Aspartyl M~.lh~ ' us~ in E. coli The plasmid pBluescript SK(-) (pDM2: Genebank accession # S37495) GontainS the entire coding region (SEQ
30 ID NO:9) for the morç acidic isozyme ll of the human L-;~ud~lual~ D-aspartyMll~ llldllsleld~e (SEQ ID NO:9) (MacLaren, et al. (1992) ~iochem. Rioph~/s.Res.Commun. 185:277-283.). This plasmid is obtained from a cDNA
library derived from HUMAN brain tissue (Stratagene, La Jolla, CA) using a mouse cDNA as a probe. As an a~;on vector, a plasmid containing cDNA encoding isozyme l(SEQ ID NO:1) or isozyme ll (SEQ ID N0:2) having se~ nres other than pDM2 (SEQ ID NO:9) can be used to produce isozyme I having the sequence indicated as SEQ
35 ID N0:3 or isozyme ll having the sequence indicated as SEQ ID NO:4 so that each isozyme can be produced in at least ei9ht forms (l22l119K205 l22l119R205~ l22V119K205~ l22Vl1gR205~ L22l119K205~ L22l119R205 L22V119K205 CA 0220326~ 1997-04-21 Wo 96/12797 PCT/US95113691 L22V11gR205) as described earlier. All~ ,'y, isozyme lll with a R C-terminus (226 residue long) can be produced in the same manner. Such plasmids include pBK phaye YeCtor (SLId'L~, Jf) and the pTrc99A !,~ plasmid (PhallllaL;a, f~ ,al~ ay, NJ). E co/iis preferred for expression of human methyltransferase because tl~alaLI~.
of iso~yme ll purified from human elyIhl~ s has shown that it is not pOst-ll~ modified (Ingrosso, et al. (1989) ,/Ri~.~Clr 264:20131-20139.). Thus, the E. coa expression system can produce the recombinant mbIh~ d"~r~,d~ nearly identical to the human enzyme. The pDM2 plasmid already contains a T7 promoter site in the proper position and u, for ~ , of the insert cDNA but does not contain a ribosomal bindinQ
site (MacLaren, et al. (1992) Biochem. Biophpl/s.~esCommun. 185:277-283.). Thus, the pDM2 plasmid is modified to give the u~ A,u,e~s;un vector, pDM2x, by replacing the region between the T7-promoter site and the start codon of the enzyme with a synthetic fragment containing a strong ribosomal binding site (Hine, et al. (1974) Proc.ll/atl.Acad.Sci. USA 71:1342-1346.) (Fig. 1). In Fig. 1, the Kpnl//ar1 fragment from pDM2 is replaced with a synthetic linker c - v multiple cloning sites, a eukaryotic initiator site, and a stron~ ribosomal binding site (Fig.
2). The fragment shown in Fig. 2 was D - dl~d by ,~ o~dae chain reaction (PCR) using ~ ,Jr ' lill~ primers ~u"i 3 the Kpnl and /~/8rl l~ DL.liUn sites for proper insertion. The regions 5' from the Kpnl ,e;,l,i"l;un site, including the T7, .u,,,uL~, site, were originally part of the rn' - i,,l SK(-) plasmid. The -~9 -es 5' of the ~Icol start are not present in the original l"t:lhtlll ~,ase cDNA insert. pDM2 is a jf~lu ..Lli,UI SK(-) tSIIdl~ ll.,) plasmid containing the human l"~lh~ n..fe,d~e isozyme ll cDNA inserted into its EcoRI sites. Fragment sizes given exclude the Kpnl and /I/arl 4 and 2 base-pairs U~G~ . This " ,' - I fragment has been: ~ - Ld to also possess multiple cloning sites for the insertion the Ill~lh~llldll;~f~ld~e cDNA (Fig. 3) into different ~A~.,essiùn 20 systems and û,ya,,;~.,,s. In Fig. 3, the EcoRI site at the 3'.end of the sequence shows 7 bases that are part of the pBluescript SK(-) cloning plasmid. The regions 5' from the Kpnl ,~ .I,i,,i ~, site, including the T7-promoter site, are part of the pBluescript SK(-) plasmid also. The fragment has the sequence SEQ ID NO:8. The ~urli,le and llall~6lad amino acid sequence of the pDM2x LAul plasmid from the T7 promoter site to the 3' EcoRI linker on the tail of the human cDNA insert is shown in Fig. 3. The structure of the modified region of pDM2x and the 25 overall plasmid structure have been cu"R, ' by DNA sequence analysis and reSLli~.liu:l n d ' digest analysis, r~s~uEclNLly. Any synthetic polylinker containing ribosome binding and initiator sites capable of being inserted into a Ill~lh~lll....~f~ld .E encoding vector are within the scope of the present invention. The T7 RNA pûl~"" ,dse-driven eA,u~ ~5;UIl plasmid is then 1, 'e~.led into E. colistrain BL21(DE3) (Studier, et al. (1986) J. MDL Biol. 189:113 130) for eA~ul~s ,;ùll of the human L-i~Ga~pd, lyllD-aspartyl ""~ ,a"sfelase. This strain of bacteria contains a phage T7 30 RNA pul~lllela~e yene in the c~"".. asu",a under the control of the isopropyl~ D-i' ;, ' ~u~u~dnG~;dd(lPTG)-inducible lacUV5 promoter. Other bacterial host strains c,, J an IPTG inducible T7 pcl~",~,a,e gene are also ccnl~"~.lal~d. For example, it is possible to use a dual plasmid system where the T7 pul~. ase is encoded behind a heat-inducible promoter on plasmid pGP1 2. This plasmid can be l,a"~ ""ed into a variety of bacterial strains.
~rl' ~ L...;.e P, if;
The initial batch ,u.,,ificaliùn steps used here to enrich the ",~ ar,~ d .e fraction in the Iysed bacterial extract is a modification of the p,oc6d~ used by Fu, et ~/. (Fu, et al. (1991) ,/R;7/Ch~m 266:14562-14572.).

CA 0220326~ 1997-04-21 After Iysis of the overexpressing bacteria by sonication, the cellular debris is removed by centrifugation. The nucleic acids remaining in the ~ alalll are removed by addition of a I ~ ~i, t, I"ui sulfate or pcl~lh,l~
and ~,e~ dliûm The amount of protamine sulfate required is optimized, and it is found that addition of 0.1 volumes of a 4% solution of ,.,ui sulfate gives a good F i" of . :hyll, ~Llaae in ejs ..i 'l~
5 quantitative yield. The methyltransferase then is CoilLl~ all~d and purified further by precipitation with ammonium sulfate. Again, c 'it for this step in preliminary , i la are optimized, and it is found that although the best p~ fil~alion occurs between 50 to 55% saturation, the best compromise between yield and I i~i~.ai occurs at 60% 5al~. ' and these c ' ~ s are used in the present large-scale purification. The pelleted protein is then nl ' ' in a small volume of buffer A and dialyzed against buffer A to remove the sulfate.
Then a rapid, one-step column p~l.c~ 'L.l: for purifying the Ill~lh jltldll~i~id~ from the dialysate by using DEAE cellulose anion exchange ~.h~ulllai O , ', is ~ ~" 3d It is found that 3,, " - of ll ' ', :' -'-' " ~
where the enzyme was bound to a column in a noninteracting cationic buffer resulted in incomplete ~ J,iii~.dliu".
However, the use of an ;"l~. ~ anionic buffer under ~ "' iu,., o--' - was found to result in the isocratic elution ûf homogenous enzyme. If the chromatography is ~ ' where the column is fully e~ '' dled before 15 loading, then the Illell,,lLl '~,ase is only slightly retarded and elutes close to the void volume along with small amounts of contaminating pDb~l, liJ~,s (data not shown).
'' ' - of L-lsoaspartyl Methyltransferase in Plants Peptide-d~p~..d~,..l L-;aoaspal~yl, ' ~ILIdl,ar~,aae is found in the ~ _lalN~ cells of the green alga C.
Sal~l~ii, demonstratiny its presence in at least one species in the Kingdom Protista. In the Kingdom Plantae, ~ hylL,a,,a~làae activity is detected in both classes of the a. D ~ , the monocots and the dicots. The level of activity in different tissues varies ~, ' ."y. Of the species assayed, the highest specific activity of the hyl~ Idse is found in wheat embryos (germ). In contrast, almost no .I~L~ '' L-;aOaa~udl lyl peptide-specific hyllla"ar~,aae activity is found in the leaves of lettuce or the fruits of tomato. The specific activity of the enzyme in wheat germ ~14.0 pmollminlmg) au".a;,ses the levels found in E. coli (1-2.5 r 11~ ' !mll Fu, et al. 1991) and human erythrocytes (1.9 9.4 ~ Jmg- Ota, et al., 1988; Gilbert, et al. 1988). Thus, wheat germ, a cheap and abundant byproduct of wheat flour ~JIudL~.liull, is an excellent source of material for enzyme purification."
L~ l r -~ U..L in Wheat Seeds and S " ~, Peptidc dE,.~,.,d~ L-iaoa~d~ l~l ",~lhylt~ laae aGtivity is highest in mature wheat seeds and the activity is a;y~dRI,allll~ reduced following imbibition and b ~ ?liun. Northern analysis shows that Illt lhtlllalla,r~ldae mRNA
30 is e,~ ssed as a single 1ZOO ,.,.~.lr,~,li '~, species only in seeds, and not in whole seedlings, leaves, or roots. The levels of the enzyme vary depending on stages of caryopsis db~!op,l,~"l, and the highest level of ",~ll,yll,d~ ,dse mRNA is detected in stage IV seeds whose embryos have reached maximal size while no ",~O,yll~dl,a~,dse mRNA
is d~ lable at stage ll. Northern analysis shows that 10 h ll~aln~ la of water d~ l,y, exposure to 50 ,uM
(+)-cis,trans-abscisic acid) (ABA), and exposure to salt stress (0.25 M NaCI) Jlanldli,~ induce the ~ Jr~aa;un of 35 Ill~lhyllldl,al~,dae mRNA in wheat seedlings. In contrast, Illt:lhyl~ ldse gene e~pl~aa;oll is not induced in seedlings exposed to low (4C) or high (37C) l~",pe,dl,.,e stress. These results indicate that the induction of L-.

CA 0220326~ 1997-04-21 W O 96/12797 PCTrUS95/13691 isoaspartyl ,nOIl,~ d~ O,I.se mRNA OU.~ - and ollLr li,, activity occurs not only in seed d .~L, I and 9~ , but can also be , ~, ' t~,~ in seedlings during periods of water deficit and salt stress. In particular, when the seedlin~s are treated with both ABA and NaCI, hrltl ~OIaae gene e Os;~;ùn is increased 1 1~ two-fold over the effect of either agent alone. The additive effect of a combined ABA-NaCl D~Oall...,..l 5 su~qgests that the methyltransferase gene may be a salt~ qene in addition to an ABA-. ~ gene. The hormonal and E..~ ' ' stress r._ce~sa,~ for inducing e, . of ~ :hylll Cu,a~e is ~.r~O~ Iy an ABA
collcellll of 10-100 ,uM, a salt c~ncO"I,dlidn of 0.1-1 M, or a dehydration time of 5-24 hours (Although d~ rdu may be lar~er for other plants up to 7 days).
F ir of L~ rl Methtlt~_ ' u~ from Wheat Germ Because of its high mrlhyll~ s~e~a~e activity, the wheat system was chosen. The present p~.liri.,dliu., strategy is based on the partial "~.ir of the protein carbûxyl mrlh~ a"~o~dse reported by Trivedi, et al.
(Trivedi, et al. ~1g82) Eur. J. ~iochem. 128, 349 354). ~L :h~ltl 'e.a ,e is purified from a cytosolic fraction of raw wheat germ. This material is first rrd~ d by DEAE-cellulose ~h~ alG~aphy at pH 7-10, r rrOIdLly at 9.3 (Fig. 12A). Active fractions are then saturated to 60-100%, I~IO~OI Sl~ to 80%, with sulfate in the 15 presence of â protein carrier such as Celite 545, poured into a column, and D lud by reverse sulfate yradient solubilization at room ~ lul~ (Fig. 12B). Active fractions containing a~,u,u,.illldl~!y 20 50%, preBO. ~
26 31% saturated ammonium sulfate, can be further purified, o~ol~.bly by using Sephacryl S-200 gel filtration ch~ulllaluyl h~, although other gel filtration materials are also contemplated. Surprisingly, the L-isoaspartyl methyltransferase elutes in a highly purified state in a fraction neerly t~..lL_,O-' _ to the total vclume of the 20 column. This step is unique in that the II,~lhrll- IOIa:~e iS not lldL~ on the basis of its size. Rather it is ,..ygO~Iad that the Illoll~llr 'e,..;,e asscc;dl~;, with the Sephacryl S-200 resin through Iryd~lr' 1~ hllOIa.,liuns due to â solvent effect created by the relatively hiyh c lldt lr of - sulfate in the fractions (Belew, et al. (1978) J. Ch~unldluyl. 147, 205-212). In the absence of ammonium sulfate, the nlOIh~llldll~OIdse elutes from the Sephacryl S 200 column in a position c~ , .l with a . i~ molecular weight, alony with numerous 25 co" li..~ POI~ OS~ Thus, the ~uccos~lul isolation of a highly purified enzyme ull,~..d6un from the gel filtration column is allriLuled to this unusual àbSul,ul;a~. r~
This pr,l~ li.le r I--r r ' to the L-i;,oa.,udll~N..Olh~ ld~e as assessed by ~ 9 individual gel slices in the presence of Triton X-100 as described by Clarke IClarke ~1981) Biochim. Biophys. Acta 670, 195-202).
The purity of this p.euaraliui. can be as high as 80 100%, estimated from der.;,itl Iry of the Coomassie stained - 30 gel (Fig. 13).
DNA Se7 e of the Gene '~ Ll ~ IIDe, l~ ' u~6 from Wheat The DNA sequence of the 952 bp cDNA insert in the plasmid pMBM1 is determined using the se4ur",; ,9 strategy shown in Fig. 14. The DNA sequence of the nllrllrdrl ,leldse cDNA and its deduced amino acid sequence are indicated as SEQ ID NOs:5 and 6, rO~Ecl;.Oly. The ce' ' l~d molecular weight of the 230 amino acid poly,uO,uli '~ deduced for the 690-bp open reading frame is 24,710. In contrâst, purified l"O0"dlla"~1O,ase migrated as a 28,000 Da poly,ueutide as dololl ,ed by SDS pGl~acly; ' gel elOclrupholO~;~ ISDS PAGE).

CA 0220326~ 1997-04-21 WO 96/12797 PCT~US9~/13691 C~ , i of S-, ~I Peptide rr " of L~ ,l ' tlL~ e from Wheat Germ and Its Predicted Amino Acid Sequence from pMBM1 IUIL.~ r ' at 12 sites between the predicted amino acid sequence of the wheat cDNA and the sequence of the peptide llay."~ of the wheat germ Li~ùa~Ja~ m~lh~ltla"~ .dse are found (Fig. 15). In 5 six of these positions, the ! . Ially determined amino acid sequence data clearly show the presence of an amino acid not encoded by the cDNA. At the other six positions, residues in addition to the encoded residue are identified by Edman d~y.ddaliùn. These results are cun~ ..l with the hexaploid nature of this species of wheat, where the three diploid ~enomes (AABBDD~ can contain alleles with variant s , leading to the I, ' of variant gene products ~Peumans, et al., (1982) Plant~ 154, 562-567 and Wright, et al. ~1989) J. Mol. Evol 28, 327-336). Most 10 of the amino acid changes are located outside of the three highly c ~d regions shared among r"~ tlt~ ~ferases. It is hll~lt.~lilly to speculate that these amino acid differences can result in enzymes having slightly different methyl acceptor speLi~iLi~ , which would give the cell the ability to recognize and ,c~t~ repair a wider range of damaged proteins. Polymorphisms in the human m~lh~lllall~ld~ gene have also been identified (Ingrosso, et al. (1989) J. ~ioL Chem. 264, 20131-20139 and MaGLaren, et al. (1992) Biochem. Bioph~s. ~es.
Comman. 185, 277-283).
~, ' - of Isolated ~ Wheat Germ L- so . l11 Protein Methtll, ' ..~6 The Ill~lh~llla~ la~e cDNA insert is inserted into well-known I uka~yuliL ~ du~ vectors as described for the human enzyme and used to llàn~l,lll Lulll" l~lll E. coli, followed by induction of the T7 pG~ la~e gene with IPTG. The L~ a~;un of the plant enzyme can be done in exactly the same manner as the human enzyme.
20 The bacterial e.~ ;,;u.. system is designed to express cDNA se, ,r- I~ yal~" of their phylLO : origin. The ~A~ -~;,scd r. ' : protein is purified as described above.
Ap, " for L-i ~ua~Jal l11 Meth~ ,.u..~
'' :h,lll ~ferase catalyzes the S ad ~lu. ;' aine dL,. ad~..l ...~lh~6liun of atypical L-;~oa~pa, lyl and D-aspartyl residues in peptides and proteins. This reaction can not only be used as an analytical tool to detect the 25 presence of these altered residues in aged and stressed proteins, but can also initiate a non ~ y.-,dliL pathway that can result in the rvr~a.~;on of these residues to normal L-aspartyl residues.
The ",~lh~llldu~l~,ase enzymes of the present invention can be used in cu....eLIiun with the d~ liun of L-;~oasl.d,l~l~ and D-aspartyl residues in peptides as disclosed in U.S. Patent No. 5,273,886 to Aswad, ;IILGl,uGldl~d herein by the previous reference thereto above. Briefly, this method involves breaking the poly,u "liJe 30 into ~laulll ~ using a ,GIUIeOI~I;L enzyme and then qud"lilali. '~ Lhylalillg the ;soaspa,lyl residues in the Ddylll~ using a Ill~:lhylllall,~ld~e enzyme. The total amount of methyl groups hlLGr~J~Idled into the lldylll~
is an indication of the amount of ;aGd~ual lyl residues in the pGl~e,uli-le. The amount of ;~oâs,ua~ Iyl residues in the poly,ue~,lide can be used as an indication of the amount of damage to proteins, such as those used in II,~,ap_.~liL
arpli aliulls.
I"lel~li"yl~, the structure of the recombinant enzyme is different from that of the purified enzyme from human c.yll~ûe~rles at the N-terminal alanine residue, and, as d~l~,lll;"cd by ~leLI,osprdy mass ~,uecl~uscopy, the CA 0220326~ 1997-04-21 WO 96/12797 PCT~US9a/13691 I~Lr~ h' - L enzyme does not contain covalent pOSt-tr ~ a" Thus this enzyme is suitable for use in human studies without the potential problem of antigenicity. Because the endogenous human ~ t:.yllla~ la ,e is limited to the cytosol IClarke, S. (1985~ Annu Rev Biochem 54:479-506), damaged proteins in the . ,.I._ " ' ~ ...;., cannot undergo repair catalyzed by this enzyme. Thus, injectable and topical therapeutic preparations 5 using the methyltransferase and its substrate S-adenosylmethionine are useful. Since the IL ' I human enzyme has no potential problem of - ~iu ~" it may be injected directly into the brain, eye, blood stream and so forth.
In addition, purified plant enzyme can be used in skin-care products as a topica! ".,, _ since it also ,~ 1l, !S
damaged ;~a~,ua, lyl residues in peptides and proteins.
By . ' ~-il,d to a tissue an amount of methyltransferase, preferably in conjunction with its substrate 10 S-ad~nG;,yll"~lhionine, sufficient to convert said L-isoaspartyllD-aspartyl residues to L-aspartyl residues in the tissue, ai ' for a medical condition ~ n, d with an increase in L-isoaspartyllD-aspartyl residues of poiypeptides in a tissue can be performed. Such medical C~ or- include those resulting from crl s ' ' ., of matrix proteins and dc~, dda of flexibility of skin tissues such as cataracts, Alzheimer's disease and the like. For this purpose, either the human or plant enzymes can be used, and the dosage of the enzyme is such that the co"~..l-al;un of the enzyme in the Pl-r d-' iS in the range of 0.4-40,uM. The enzyme can be r~ h6l~d simply in the form of an ointment with S-a.l~"us~l~"~ ionine and a ~Jha,."ac,,..lil.dlly âcceptable carrier. A typical ointment can contain the enzyme in an amount of 0.D01-10% by weight and S r ' ~ yln ' B in an amount of 0.00004-0.4% by weight.
Other medical c~ lil are formation of plaque in brain tissues and degradation of cellular function in brain tissues, and, for these purposes, human enzyme is pl~ dti~ used in an amount such that the cull...llldlion of the enzyme in the ~ nl" ~ space is in the range of 0.4-40 ,uM. For ~aIiùll to the brain, the enzyme can be provided as an injectable solution typically co.,i ~ the enzyme in an amount of 0.001-10% by weight and S
ade,iûsyl~ in an amount of 0.00004 0.4% by weight in a ~ha""d~,e.,i 'l~ al.c~ carrier. Preliminary evidence suggests that L-i~oa~,ud,lyl and D-aspartyl residues can ' le in the amyloid protein of Alzheimer's disease. Since a fraction of ,~-amyloid protein is found in the ce,~hr sr ' fluid (CSF~, it may also be possible to treat Alzheimer's disease by injecting the enzyme into the CSF.
Another medical condition is dey~ d of flexibility in a vascular system, and, for this purpose, human enzyme is ~ ldbl'r used in an amount such that the t~ ~ llaliùn of the enzyme in an e,yIh,.ry~ ndo;' " ' tissue, coronary artery tissue, immune cells, receptors of âll cells or lungs is in the range of 0.4-40 ,uM. For the vascular system, the enzyme can be provided as an injectable ;Illld~nous solution typically cu,.i ~ the enzyme in an amount of 0.001-10% by weight and S e~ 1~ yl~"~lllionine in an amount of 0.00004-0.4% by weight in a phd""ac~u -'Iy acce~Ji '' carrier. The solution can be ? ' ' d~ d by means of a catheter or direct injectiûn.
Other medical coru;li ns are infertility related to eggs andlor sperm and formation of fibrosis in tissues, and, for these purposes, human enzyme is p,~,dbly used in an amount such that the conc~"lrdlion of the enzyme in egg or sperm cells is in the range of 0.440 ,uM. For the vascular system, the enzyme can be provided as an injectable solution typically c~r.IL;.. ;.. g the enzyme in an amount of 0.001-10% by weight and S ade.. osyl.. ~i' .e in an amount of 0.00004-0.4% by weight with a phdllllactu~ Iy ar,,~,.i '' carrier.

CA 0220326~ 1997-04-21 Since L-;~oa~,ud,l~l and D aspartyl residues are aL~.,A.dlL'~ recognized by methyltransferase, it is possible to d~the presence of these damaged residues in, ' - ' ,Gel~, ,Jt;dds so that the purity and shelf-life of such protein products can be verified. These assays are performed by incubating the pharmaceutical preparation with Se~ yl [14C-meth~ .,. i' in the presence of the purified ~ lh,d~l -f~.a,e and d , g the 5 1 ' ~eli.i~y llall~r~ll.,d to the pha~ ..R ~I This is done by incubating the reaction products with an alkaline solution to release bound methyl esters as radioactive methanol, which is then collected in scintillation fluid as described (Lowenson, et al. (1991) J. BioG Chem. Z66:19396 194061.
Further, diagnosis of disease states in which L-;~ùd~,ua,lyl and D-aspartyl residues accumulate may be pC.~"I.. d by I - b~o the content of L-i,ùaO~.a,lyl and D-aspartyl residues -~[ ' ' in a disease acsoi:3lrd 10 protein, by using methyltransferase as a probe. Since a fraction of ,~ amyloid protein is found in the cerebrospinal fluid (CSF), it is possible to develop a :' ~ - test for Al' 's disease by j"GO the content of L-;~oa~pd,l~l and D-aspartyl residues in samples of CSF. It has not hitherto been possible to obtain an accurate diagnosis of this disease which ~ paN~ O millions of Americans. The 1 test can be ae- , " ' -d using the same assay described above for protein l~h~llllaC~...Ii~.al quality control.EXPERIMENT 1: NUCLEOTIDE SEQUENCE OF HUMAN METHYLTRANSFERASE CODING REGION
cDNA Library Synthesis and Clone Sl.- _ A cDNA library cù"~ll.,Ll~d from the temporal cortex of the brain of a 2-year-old female human was ~,u.uhdsed from Stldldyt:lld (ff~352~l. The cDNA was ,~"lh_~i~dd from oligo-dT isolated mRNA, and packaged into the EcoRI sites of the lambda ZAP bacl~,iu~hage vector (Slldi " e) The library was ~ 9 ~dd in E. coO BB4 and 22 plates c."i 5 5 x 105 plaques each (1.1 x 107 plaques total) were screened using a, " '~ 769 bp H~elll fragment from the coding region of a 1580 bp murine 1llt Ih;lL,ar"l~,ase cDNA (Romanik, et al. (1992) Gene, 118:217 222). The fragment was labelled with [a 32P] dCTP to a specific activity of 109 cpml,ug with the PRIME-IT random priming kit (S~,dlay~"e). Standard plaque lift and Southern blot p,.c-' t:5 (Sambrook, et al.
(1989) '1(' ' Cloning: A Labc.dlo(y Manual, 2nd Ed., Cold Spring Harbor Labo,dl~.y) produced three positive signals. The clones for these plaques were isolated by s--'-e~ sc,~ ~ The clones were repacl~ged into plasmids in XL1-Blue cells via in vivo excision according to the ~IZAP protocol. Successful excision was denoted by ampicillin r~ Lnce. The cells cu"i O the insert-carrying plasmids of interest were grown in LBlAmpicillin medium, and their plasmids isolated and purified using Qiagen plasmid isolation columns.
N ' I 'l Se, -e DLt~.l ' and Analysis The ~ uli~lese4~ ces of the clones were r!~l~, cd on both strands by the dideoxy chain-terminating method (Sanger, et al. (1977) Proc. Natl. Acad. Sci. USA 74:5463 5467) using the Seq ~ e 2.0 kit (USB), M13 and T7 universal primers, and sy"ll,e ,;~d 22mer primers. The sequence data were analyzed with DNAStar programs on a '1e lu~h computer.
Three clones out of 1.1 x 107 plaques gave a positive signal and were isolated. The seu,.~P~l~es of two of the clones (pDM2 ~SEQ ID NO:9) and pRK1 (SEQ ID NO:10)) were .I~I~"";"ed from both strands. The plasmid pDM2 is available from I~PnPhanl~ under accession # S37495. In SEQ ID NOs:3, 4 and 9, the encoded initiator CA 0220326~ 1997-04-21 W O 96/12797 PCTrUS9S/13691 methionine is numbered O and the next amino acid, alanine, is numbered one. This is done to match the numbering scheme of the final protein due to the excision of the initiator methionine. Numbering for both clones begins at these positions. Clone pRK1 begins at position 358. Numbers to the left of the divisor represent pDM2. Those to the right of the divisor are for pRI~1. The ~ . and encoded amino acid sequences of pDM2 is shown under (al 5 and continues to (b). (b) IL~,.t~ the 47 base insert found in clone pRK1.
EXPERIMENT 2: HUMAN METHYLTRANSFERASE tXrHt~SIOhl IN E COW
Cun~ll. of expression vector pDM2x The pDM2 plasmid was modified to giYe the u~ , I vector, pDM2x, by replacing the re~qion between the T7,..~ le, site and the start codon of the enz,vmé with a synthetic fragment containing a strong ribosomal 10 binding site, as IJIG.- ~Y described (Figs. 1-3).
~~ I ial Growth E. coli strain DH5~ IGibco-BRL, C- ' L ~, MD) was used for cloning and, opa~Oliun of plasmid co.,.l"..,l~. Tra"~r~,llla6uil of ~. coD was acc ,' '1~ ' by the one-step method described by Chung, et al.
(Proc.lll~tl.Ac~d.Sci. USA ~19~9J 86:2172-519). For protein e,~u,~s;ul,, E coli strain BL21(DE3) (Studier, et al.
.1.~ ;ql(1986) 189:113-130) was l~ rl,.,.. dwiththepDM2x ~ ;u,lplasmid. BL21(DE3)bacteriacontaining the pDM2x plasmid were grown at 37C in LuriaBQ~lld"; (LB) broth (Sambrook, et al. "Molecular cloning: a Idbolàl~ manual," (1989) Cold Spring Harbor L ' a; y, Cold Spring Harbor, NY) medium ! I 5 1OO ,u~qlml .
Protein Concentration Determination Protein Cullc~ 'alions of crude extracts were d~: d by the l, ' ' uaL~IiL acid-Lowry method (Chang, Y.C. (1992) ~n:~lRjn~/70m 205:22-26) with bovine serum albumin as a standard. Protein cllnL~:Illldliuns of column fractions were d~ r' by ...~,a~ g the optical density at 280 nm and equating an ~ I,a,..,e of 1 to a conc6rlllaliun of 1.0 mglml for a mixture of proteins (Sambrook, et al.(1989) "Molecular cloning: a lal,ordlû,y manual," Cold Spring Harbor I . 'D~dluly, Cold Spring Harbor, N.Y.) or 1.12 mglml for ~ ~ - r..~lh~ a"~r~,dae (Mach, et al. dnol~7;7,~ (1992) 200:74-80).
Meth~ . Assay The cunc~ of active ",~ l0a~e,aae was d~ d by P i~y baselabile methyl ester ~ulllldliun on the methylacceptor G.," using a vapor diffusion assay IGilbert, et al. ~j",~'r ~ 19~oY
27:5227-5233). Final conc~"l~dlio..s in a 50 ~L reaction mixture were 10,uM S a~' -syl L-[methv/14C]I"~i' ~
(53 mCi,'n ', 100 cpmlpmol, ICN ~i ' Is, Irvine, CA), 40 mglml chicken ov " Ifraction V, Sigma, St.
Louis, MO), and 0.2 M sodium citrate, pH 6Ø I,,cubaliu.ls were done at 37C for 30 min and quenched by the addition of an equal volume (50 ~L) of 0.2 N NaOH, 1.0% (wlv) SDS. This mixture was spotted on a 1 cm x 9 cm piece of thick filter paper (No. 165-090, Bio-Rad, Richmond, CA) prefolded 1n an accordion pleat and placed in the neck of a 20 ml plastic sc;" 'l;liun vial co"i 9 6 ml of SafetySolve counting fluor (No. 111177, Research Products h,lLr,,dtiuaal). The vials were capped and, after 2 hours of incubation at 23C, the filter paper inserts were removed and the vials recapped. R. ' - ~;.ity was measured over a wide 14C channel. Once purified to CA 0220326~ 1997-04-21 Wo 96/12797 PCT/US95/13691 hon ~O :y, as determined by SDS-PAGE, the specific activity of methyltransferase was measured ~10,000 rng at 37C). This value was used to dtl~. , h~lt~ ~la.~e mass from enzyme activity IlladaUI1~ u~l3~
~r ' ~ ' u~e Expression Ten one liter cultures of E co/istrain BL21(DE3) containing ~IJIea~;ull plasmid pDM2x were grown at 37C
in LB broth containing 100 IJglml ampicillin with shaking at 250 rpm to an optical density of 0.5 at 600 nm.
Ex~ of the enzyme was then induced by adding IPTG to 50 ~M. Cell growth was allowed to continue for

4 more hours until growth reached saturation at an optical density at 600 nm of 1.6. The cells were hdl~ d by c~..l,i~, valiu~. at 5,000 9 for 15 min at 4C, yielding a 14 gram wet weight pellet. All a,. ..: from here until 10 the column fractionation were done at 4C.
To d~ - the optimal co...,~..l. of IPTG for . t:~;u~ log phase E~ aa;uu cultures were induced with a wide coac~lllldli~ll range of IPTG and assayed for methyltransferase concentration. Fig. 4 shows that only very low amounts 10.03 mM) of IPTG were required to induce EA~ ~ of the l~I~O~ o.ase. In Fig. 4, a 1 liter solution of LB broth at 37C containing ampicillin at 100 ~glml was ;--GGuldl~d with BL21tDE3) E. co/iharboring the pDM2x Ill~lllyllrall~ldae ~ ,-- plasmid and grown to an optical density of 0.2 at 600 nm. The culture was divided into 4 equal volumes ~250 ml each) and brought to final conc~..lrdi of 0, 0.03 mM, 0.50 mM, 04 8.00 mM isopropyl ~ D thiogdldblu~ dnoside. Vigorous shaking at 37C was continued and samples were collected over 3 hours. Each sample was placed on ice and the cells were pelleted by low speed c~lllriruydliull. The pelleted cells were l~sv~lJrllded in buffer A, sonicated, and assayed for I~ hyltld~ ldse activity. The c~"..~"l,dlion of soluble protein was d~e" -~ by the modified Lowry assay. A specific activity of 10,000, ~ rg was used to calculate ul~ rdlla~eldae COu~ rdliull. hll,uû~ , the ul~ llldll~eldse was found to comprise up to 20%
of soluble total protein in fully active form and, as determined by SDS-PAGE, no ..._lhyllrdll ,~eld~e pul~ ,Dde was found in the insoluble fraction where inclusion bodies are usually found.
A more extensive analysis of Ih~ -d~e r udùl~liull using a conc"..l~di of IPTG of 50,uM was 25 then p~ ..",ed. The data in Fig. 5 show that enzyme ~., ' liu" occurred rapidly during log phase growth, resulting in an i","~a;,;"y ac~.u,,,uldliu,, of nl~ llrdll~ld~e. In Fig. 5, BL21(DE3) cells cOIl ~ pDM2x were cultured in 1 liter of LB broth at 37C with shaking at 250 rpm. Induction of ~A~ aa;ull~ begins at time zero by the addition of IPTG to 50 ,uM. Soluble protein and Ill~lh~llrdll~ ldse were assayed as for the times shown in Fig. 4. Only when cell growth began to slow down did the fraction of Ill~:lhrllrdlls~elda~ in the soluble protein fraction level off.
30 EXPERIMENT 3: HUMAN METHYLTRANSFERASE PURIFICATION
Me~lh~ rL..u.l~! PL.iri.,u;
Buffer A was used ll". ~' - I all the ~u~epa~dliùns and contained 5 mM sodium phosuhdle, pH 8.0, 5 mM
EDTA, 25 ,uM I ' ~Im~lhd~aul~u~l fluoride (PMSF), 0.1 mM ,' ' 0ll~;lol (DTT), and 10% vlv glycerol.
The cell pellet (14 9) was c~ e"rlPd in 200 ml of buffer A at 4C. The bacterial cells were Iysed by SOI 0un for 2 min on power level 5 at CO"Ii",JùUs output using a Branson W-350 sonifier with microtip probe.
The sc 1tiOil was done in a 500 ml beaker cooled in an ice water slurry bath to dissipate heat buildup and in a CA 0220326~ 1997-04-21 W O96/12797 PCTAUS9~/13691 . 15 manner ensuring that thorough mixing took place. These - - settings were optimized in p,~' y ~, : where samples were Iysed at various power levels for various times and assayed for enzyme content by ,,,~h~lbd,,~rc.d~e activity. Cell disruption began at power level 3, while activity of the enzyme began to decrease at level 7, ~... "y due to ov~ alil,u. The Iysed cells were c.,..l-irl" ~ at 13,000 y for 15 min and the

5 supernatant saved. 20~ ml of buffer A was added to the pelleted debris and this material was again ~ d, pelleted, and the au,u~ aldul saved. The ~.,u_. : of the two extracts were combined to give a final volume of 390 ml.
- Nucleic acids were ,u,e~;~,it~l, d by slow addition of ".u - sulfate (0.1 volumes (39 ml) of a 4% wlv , ui sulfate solution (Grade X, Sigma)) at 4C with mixin~ to the combined supernatant fraction. After mixing 10 for 30 min, the solution was .. ~ ed at 13,000 9 for 15 min and the ~uu .. - saved. The Ill~lh11lldll~reldae was con~ ~,dlud and further purified by slowly adding solid ammonium sulfate (Ultrapure, ICN) to 60% saturation (167 9) at 4C, mixing for 30 min, and centrifuging at 13,000 9 for 15 min (Scopes, R.K. (1993) "Protein Hti~.dliull. principles and practice," Springer-Verlag, New York). To remove the ammonium sulfate for the ,eu~u~l anion-exchange column ,uuli~il.dliùn, the protein pellet was l~e~ e !~ ' in 18 ml of buffer A and placed in a 3500 molecular weight cut-off dialysis u~r",L,dna (Spectrapor 3, Spectrum) and dialyzed three times, every 12 hours, against one liter changes of buffer A.
The final ~-u.i~i.,dLun step used DEAE-cellulose ch.. : ~ d~ under ! , '' C~r ~it;un~ at room l~ld,u~ldlL.~. A 14.7 cm high x 2.5 cm l.D. DE-52 (~ ldluldll) column was equilibrated at a flow rate of 2 mllmin with buffer A as determined by ~"ea ,u,;"g the pH of the eluate. In preparation for sample loading, the column was 20 washed with buffer A with NaCI added to a final culllellll_' of 1 M at a flow rate of 2 mllmin for 1 h, and finally washed with buffer A in the absence of NaCI for 6 hours. 3 ml of the dialyzed sulfate ,ul~?dldliun was then loaded onto the column and washed at 2 mllmin with buffer A for 2 hours. Material bound to the column at this point was eluted by washing with buffer A with 1 M NaCI. 2 minute (4 ml) fractions were collected.
Edman protein ~6.,~ '~9 by Dr. Audree Fowler was done at the UCLA Protein '' us~ ,r,,Li,,9 Facility 25 using a Porton 2909E se~luen~l. [l~llua~JIdt mass :,peLl.usccpy was p~,r.,~".Ed at UCLA M-'( ' and Medical Sciences Mass Specl~u~lopy Facility by Drs. Ken Conklin and Kym Faull using a Perkin-Elmer Sciex API3 i"~lll,",~lll.
UV and visible sp~.,l,-,scopy was done on a Hewlett Packard 8452A diode array ;,,.E_LI~ Lt:l.
The initial batch puliriL~dlion steps used here to enrich the Ih~llla"~rd,d~e fraction in the Iysed bacterial extract is a ~udirildliun of the procedure used by Fu, et a/. (Fu, et al. (1991) JaiolC~SL 266:14562-14572).
30 After Iysis of the u~ ;"9 bacteria by sDril liun, the cellular debris was removed by centrifugation. The nucleic acids remaining in the supernatant were removed by addition of a ,~I~L;,U;tUIII, ~JlUi - sulfate, and c~,.l,i~uydliun. The amount of protamine sulfate required was optimized, and it was found that addition of 0.1 volumes of a 4% solution of protamine sulfate gave a 2.4-fold ~JuH~il,aliu.. of n. Ihtlllur.~re,dse in esse"lidlly u,uall6ldl;.~ yield (Fig. 6, Table 1). In Fig. 6, various volumes of a 4% wlv l.,ul sulfate in buffer A were 35 pipetted into 1.0 ml of centrifugation-cleared sonicate ~LI,U~llldldlll and vortexed. Nucleic acids and other debris were pl~L;~.ilùled at 13,000 9 for 15 min. Su,utulldldlll~ were assayed for total protein and ul~lllyllldllsr~ld~e as .. . . . ... . .. . . . .

CA 0220326~ 1997-04-21 W O96/1~797 PCT~US9S/13691 describedinFig.4.The~ lylllall~u~d~ethenwasc~r~ :dledandpurifiedfurtherby~ uit~liunwithammonium sulfate. Ayain, conditions for this step in preliminary l:A~r_9 ' were optimized. The amount of total protein and h~ltlall~rr~:ld;~e r I '," ' ~ at various, . ~ s of sulfate : .: is shown in Fig. 7. In Fi~q.
7, solid ammonium sulfate was added to 1.0 ml samples to give the indicated percent 5al~ and mixed on a rocking platform for 30 min at 4C. Protein was pelleted by 13,000 g for 15 min. Pelleted protein was resuspended in an original volume of buffer A and assayed for total protein and methyltransferase as described in Fig. 4. It was found that although the best r i~i occurred between 50 to 55qÇ ~alUldliUII, the best compromise between yield and purification occurred at 60% saturation and these conditions were used in the present large scale ~-uli~iLaliua (Table 1). The pelleted protein was then . ' ' in a small volume of buffer A and 10 dialyzed against buffer A to remove the . sulfate.
A rapid, one-step column procedure for purifying the methyltransferase from the dialysate by using DEAE-cellulose anion exchange cl..~ ' ~, a,ub~ WâS then ~ -' r ~ It was found that a,p,'il: of L,ddiliu--al methodologies where the enzyme was bound to a column in a noninteracting cationic buffer resulted in incomplete , u.i~iLnlion. However, the use of an ;llL~:ld~i' v anionic buffer under lt , '' iulll C ' was found to result 15 in the isocratic elution of homogenous enzyme (Fig. 8). In Fig. 8, protein concentration was determined by ",~a~-.,i"g the optical density at 280 nm. ~I~LIIu~hu~a;~ was p~.~....ad using the Laemmli buffer system (Laemmli, U.K.
(1970) /I/~ture Z27:680-5) using a 12% Duracryl (Millipore) s~ua,dli"y gel. r.ly~JE~ les were visualized by rapid silver staining (Blum, et al. (1987) Electrophoresis 8:9399.). Fractions were analyzed on two mini gels: 1,uL
Sonicate and Load, 10,uL of 10-21 on gel #1 and; 15 ~L of LMWS1100, 10,uL fractions 22-25, 27,29,31,33,35, 1,uL of fractions 76-80 on gel #2. Under these conditions, the ",~II,yll,an~,d~e elutes as a narrow spike of enzyme centered on fraction 18 and as a broader peak from fractions 21 to 40. The mell~yll,a"~ àse in these two peaks appears to be identical. Both peaks are d,d,dLII,.i~d by Ir~d ~ 1 25 kDa pol~ lidas in SDS gel analysis (Fig.
9). In Fig. 9, 1 ml fractions of purified enzyme were dialyzed against 20 mM sodium citrate at the pH values indicated and the fractions were ~ - : dl~d by ullld~illldli~m using Centricon-10 micro conc~"l~dlu.~, (Amicon, Beverly, MA). The first peak shows a low level of c Idlld~ldliull (less than 5% for fraction 18) by other pûl~u~,Jli.las while the second, broader peak shows no other protein c i : The specific activity of the hyllldu~ld~e is identical in both peaks, and their weight by mass ~,E.,IIu;,cu,,~ is also identical (see below).
It appears that the partial ILS-' of these peaks is due to the, lr, "' iu", nature of the chllJIlldlugldpllt as well as the amount of protein loaded. For example, a smaller loading of dialysate results in only a single broad peak of 1 ~, : enzyme eluting further from the void volume, and a larger loading results in a narrow peak with a higher cu~r,L~Irdliun of enzyme (with minor c : ~ting polyu~,ulidas) near fraction 18 (results not shown). If the Lhl L~llldluy~J~,hy is pE, ~ulllldd where the column is fully equilibrated before loading, then the Ill~lhyllldll~l d~e is only slightly retarded and elutes close to the void volume along with small amounts of CGIll ' ~Ibly poly,u~,ulides.
Table 1 ,u"""a,i~s the I~Uli~iLdliùll steps involved in obtaining large amounts of homogenous llwllltlLIdll~ldse. It shows that the largest loss in yield occurs during the - sulfate pl~L;uildlion step;
the reason for this is not understood. Overall, 17 mg of enzyme from each 1.7 liters of the original broth culture W 096/12797 PCTrUS9~/13691 at a specific activity of 10,000 pmoleslminlmg, comparable to that found for the purified human ~IyLh~ul.ytG enzyme (Gilbert, et al. (1988) ~ "~ 27:5227-5233, Ingrosso, et al. (1989) J.Biol.Chem. 264:20131-20139), was obtained. In this ,[llL~Jalaliun~ the column hl. D , h~ step using only one-sixth of the total p.-, dtiùl) (Table 1) was F iu~ ed. Repeated cycles of DEAE-cellulose chromatographycan thus be used to readily yenerate additional 5 ; 9 enzyme.
It was found that the purified recombinant enzyme is stable at room temperature for up to 2 months and repeated freeze-thaw cycles have no effect on activity. Fi Ih . ~, it was found that the "l~th~ll,d"s~G,a~e can - be heated for up to 30 min at 50C with no loss of activity.

F~ ' of human reGombinant protein-L isoaspartate (D-aspartate) O-methyltransferase from E. coli Cells from 101 of a culture in LB broth at an optical density of 1.6 at 600 nm.

Total Total Specific Purifi Sample Volume ProteinActivityActivity Yield cation mL mg pmollminpmollminlmg % fold Sonicate 400 3680.0 4800000 1304 100 1.0 Cleared S~JGIIIaldl~l 390 1358.0 3471000 2556 72 2.0 0.4% Protamine Sulfate 428 562.4 3474900 6178 72 4.7 60% A.. ,on ~
Sulfate 18 291.6 1602000 5494 33 4.2 Dialysate 18 146.0 1062000 7275 22 5.6 DEAE Pool - b Fractions 16- 220 17.2 171500 10000 21 8.0 70a a This ,L~rGse"ls the loading of 3 ml of dialysate (or 116 of the total ,ul~,ua,atiun) - the protein and l.. ~O.~ d"~e,d~e content was d~,lGII -d for individual fractions and combined to give the values reported here.
b Corrected for loading of 3 ml of the 18 ml of dialysate.
Enzyme C~ ~ tl The a\, ' ' I'Ly of a concc.,~,dl~d form of the enzyme is important for several uses including x-ray structure determination. It was found that su..ce~ ul conc~"lld6un of the Ill~lhyllld.,;,~,dse was GAllG-..~I~ pH depGI)dG..L. Contrary to typical protein solubility cha,dclG,i~liLs (Scopes, R.K. (1993) "Protein ,I-uli~i~.dliur~ principles and practice," Springer-Yerlag, New York.), cGnce.. l~dliù" of this enzyme occurred most readily near its ;~oelecl~i,, point of 5.9 pH units (M. Redinbo, personal c.", ~60n) (Fig. 8).

CA 0220326~ 1997-04-21 W 096/12797 PCTrUS9~/13691 -18~
Characteristics of Purified Recombinant Methyitransferase N-terminal , ~, of the purified methyltransferase was p"~,, ' by - ted Edman L~ analysis. The ~ r'etP~mined sequence of the first twenty residues was exactly that predicted from the cDNA with the removal of the initiator methionine as found in the human enzyme (Inyrosso, et al.(1989) .I~' 264:20131-20139) (Table 2). It was found no evidence for a blocked amino terminus, such as the d~.~lyldl~d alanine residue present in the human enzyme (Ingrosso, et aL(1989) / P~;AI rhPm 264:20131 -20139)-The molecular weight of the purified enzyme was measured at 24,551 +3 Da by .,I~ U r.dy mass s~.6clllJscG~Jr (Fig. 10 (A)). In Fi~. 10 (A), a portion of the spectrum of material purified was described above using dit' Ihlb ~( ' as the reductant in buffer A is shown. No other peaks were discerned at other molecular weights. This average value matched exactly the predicted value of 24,551 Da of the ''i~d product encoded by the cDNA ~ F~l~' y attempts at ~ i6~,dliun using ,B-mercaptoethanol instead of dithiothreitol as the reducing agent produced an enzyme that had 1 or 2 adducts of ,B ua~lu~llldllùl as indicated by the 2 additional minor forms at higher molecular weights of 24,627 and Z4,70Z Da, It~p~ (Fig. 10(B)). In Fig. 10 (B), a portion of the spectrum of material purified when 15mM ,B: L_r 6 ~ was - ' ' : ' for 0.1 mM d Lhlu;tùl in buffer A. The peak at a mass of Z4,627 l~llt~ ll i an adduct with 1,~-mercaptoethanolmolecule and the smaller peak at 24,704 rL~"~S~ the adduct with 2 ,B ,,a~.i '0 ' '~ ' The structure of the protein product by on-line liquid clllullldll,~lu~,h~ L.IIus~Jld~ mass spectral analysis of tryptic and cyanogen bromide D v : were also directly confirmed. With the exception of an insoluble hydl.,h~L core cU,l~ r. " j7 to residues 37 to 143, the mass of all detected species cor,~,uunu.,d to predicted r~dylllelll~.
For direct ~Jr,~,llU~ l,ot~ lli.. cGnc~..l,: d~ 6ull of h~ ~a IllI:Olyllldll~O~ld:~e~ the e~l;"~,i C0Lrr;L;~III of the enzyme from amino acid c ,- to cu,lt;,,uond to 1.12 mglml for 1 A280nm (Mach, et al.(199Z) An~lR--'-- 200:74-80) was calculated. This value was verified by the lli ' ' uac~6l~ acid-Lowry method using bovine serum albumin as a standard (Chang, (1992) An~lBiochem.
205:22-26). The UV spectrum of the h~ "~O~yll~d~ ,r~,ase in fraction 29 from the DEAE column (Fig. 8) is shown in Fig. 11.

.

CA 0220326~ l997-04-21 W O96/12797 ~9 PCTrUS9~/13691 .

N-terminal Edman s . v analysis of purified human .~ ' protein-L~ (D ;, I ' O - ~ from E. col;. 150 pmoles of tll~
in 10 ,uL of 10mM neutral ' ' - ' were loaded.

Residue Residue Cycle Identified pmol ~ycle Identified pmol Ala 68 11 Glu 7 2 Trp 25 12 Leu 10 3 Lys 1 Z 13 lle 6 4 Ser 8 14 His 8 Gly 19 15 Asn 6

6 Gly 23 16 Leu 9

7 Ala 20 17 Arg 9

8 Ser 6 18 Lys 4

9 His 12 19 Asn 6 Ser 6 20 Gly 13 N-terminal sequence encoded by cONA-MAWKSGGASHSELlHNLRKWG
EXPERIMENT 4: IDENTIFICATION OF METHYLTRANSFERASE IN PLANTS
E ; .
Fresh carrots, yellow corn, Romaine lettuce, green peas, white potatoes, spinach, cherry tomatoes, and alfalfa were "...~ ..;,ed at a local ' ~,ib~.lo,. Alfalfa seeds and raw wheat germ were from Rainbow Acres, Inc. (Los Angeles, CA), while soybean seeds were from Arrowhead Mills, Inc. ~Hereford, TX). Winter wheat lTriticium aestivLm cultivar Augusta) seeds were provided by Dr. Robert Forsberg of the University of \N; (Madison, Wl). Danver Half Long carrot seeds, Golden Jubilee corn seeds, Romaine lettuce seeds, sugar snap pea seeds, New Zealand spinach seeds, and Bonny Best tomato seeds were from the Chas. H. Lilly Co. (Portland, OR). A cytosolic fraction of C~ ,;,ha,~llii(Wt strain 2137) was provided by Drs. Gregg Howe and Sabeeha Merchant of the University of California at Los Angeles (Howe & Merchant, 1992).
F~., dt- 0~ Plant C~ltosol Crude cytosol was extracted from the plant tissues by h !~ liun using a mortar and pestle.
In a chilled mortar, liquid nitrogen was poured over plant tissue (typically, 20 9 of fresh tissue or 5 9 of seeds) until the tissue was c, ,'L:Iy frozen. To remove ~,I,de;,;,~b!c poly~henol oxidases pol~r,liall~
released from the tissue upon h Sv Oùn, 3 9 of hydrated PVPP (polyvinyl poly~u~nl ' 'o, ) lLoomis, et al.(1965) Fh~tl,Ll,t ",;~lly 5, 423 438) was Iho",~.yhl~ mixed with the frozen tissue before the tissue was ground with a pestle. C..l,~L.Iion buffer (20 mL of 100 mM HEPES, pH 7.5, 10 mM 2-mercaptoethanol, . .

CA 0220326~ 1997-04-21 W O96/12797 PCT~US9~/13691 1 ~m leupeptin, 1 mM PMSF, 10 mM sodium hyd~u~lllfilc, and 10 mM sodium ~ Ulrilc at 4C) was added to the mortar, and the slurry was ground further. The resulting crude ' " was pressed through four layers of ..hecac~.lulll and then cc"l,if",ed at 2200 9 for 30 min at 4C to remove the insoluble PVPP and undisrupted plant material. The resulting supernatant was r,~ ù,_c' further at 172200xg for 50 min at 4C and then filtered through two layers of Miracloth (~-" ' San Diego, CA) to remove the floating lipid layer. This fraction, identified as crude cytosol, was stored at -80C and utilized as the source of hyllldnarcldae Methylation Assay ~ Ihylllarlafc~aae activity was identified using a vapor phase diffusion assay that ~ l"c the number of, ' ' '-' ' methyl groups llallaf~llud for S ' - IlL-[methJ~ 4C] methionine to a peptide substrate by quantitating the release of [14C] methanol resulting from the hydrolysis of base labile methyl esters. In a total reaction volume of 40 ,uL, 12,uL of enzyme plcual, was incubated with 10 ,uM S-adenosyl l [methv/-14C]methionine IICN Biomedicals, 50 rCil~nnl) 500 ~M peptide substrate, and 0.33 M HEPES, pH 7.5. Peptide auLsllalcs ~VYP ~L-isoAsp) HA (SEQ ID NO:15), KASA-(L-isoAsp)-LAKY (SEQ ID
NO:16), AA (L-isoAsp)-F-NH2 (SEQ ID NO:17), VYG (D Asp)-PA (SEQ ID NO:18), and KASA-(D-Asp) LAKY (SEQ
ID NO:19)] were aylllhca;~d by Dr. Janis Young at the UCLA Peptide Synthesis Facility and chdlaclc,i~cd as described pl cv;uuad~ (Lowenson, et al.(1991 b) JR;ol chl 266, 19396-19406, (1992) J R;~ ~ChPm 267, 5985-599~). Allcll,ali.~, during the ,~ .ai ~ of the wheat germ mclhtll~ ~ 'c,aae (see below), samples were assayed in buffer cr", J a final concci,ndliun of 0.2 M sodium citrate, pH 6Ø In either case, ;1ll,ubaliuil5 were p~.fu,llldd at 25C for 60 min. Each reaction was then quenched with 40 ~L of 0.2 M NaOH and 1 % (wlv) SDS and vortexed, and a 60,uL aliquot was spotted onto a 1.5 x 8 cm pleated filter paper (Bio Rad no. 165 090) and placed in the neck of a 20-mL s,,;~,i " liun vial c~" ~, 5 mL of Bio-Safe ll (RPI, Mount Prospect, IL) counting fluor. The vials were capped, and [14C]methanol was allowed to diffuse from the paper through the vapor phase to the fluor, while the nt,,,vula61c 14C
r, ' aL~N;Iy remained on the paper. After 2 h at room Icll~ Idlulc, the paper was removed from the necks of the vials and the vials were counted.
Protein Determination A ,,,Gd;fiualiun of the Lowry procedure (Bailey (1967) Techniques in Protein Chemistry,Elsevier P Ll ' Co.,New York) was used to determine the concc,,lldliun of protein after ,ulcL;~JilOliun with 1 mL
of 10% (wlv) Il; ' ' ruaccli~ acid.
lifiualiu~ of L lsoaa~ua~ II. fu.ase in Plants Rc,ulcsc,,ldl;.cs from both classes of ~ Illa as well as a green alga were surveyed for the presence of L isoaspartyl ~ucll,ylllallafclase. Crude cytosol was isolated from different types of plant material and then assayed for l"clI,yllld"afc,dse activity using the L-isGa~uall~d-cGlll 9 peptide, VYP-(isoAsp)-HA (SEQ ID NO:15), which has been shown to be an excellent peptide substrate for the human clllhlyu~ylc methyllld"sfc,ase (Km=0.29 ,uM; Lowenson & Clarke, 1991). [nd~gc,,ù~s cytosolic CA 0220326~ 1997-04-21 W O96/12797 PCT~US9~/13691 r . t;.l~s are also potential methyl avc~"Lura~ therefore, parallel L~ ... were r ' l ' in the presence and absence of the peptide substrate (Table 3). Peptide dependent L isûdv~la~lyl m~th,dl,d,.arav-dse was found in the vegetative cells of the green alga C. I '~ J~ii, ' : V its presence in at least one species in the Kingdom Protista. In the Kingdom Plantae, methyltransferase activity was detected in - 5 both classes of the angiosperms, the monocots and the dicots. The level of activity in different tissues varied cuna;d~,dbl~. Of the species assayed, the highest specific activity of the methyltransferase was found in wheat embryos. In contrast, almost no detectable L-ivoaa,ua,Lyl peptide-specific m~lh~lt. f~,dae activity was found in the leaves of lettuce or the fruits of tomato. ~v 't; 11~, high levels of h~ Cvlave activity were found in the seeds of all plants assayed, including corn, alfalfa, lettuce, pea, spinach, and tomato, as well as in the roots of carrots and potatoes. The specific activity of the enzyme in plant seeds (0.66 14.0 pmollminlmg) is comparable to the levels found in E. OOli (1-2.5 pmollminlmg; Fu, et al., 1991) and human e,ylh,u~es (1.9 - 9.4 p !1. In v. Ota, et al., 1988; Gilbert, et al. 1988).

CA 0220326~ 1997-04-21 OCCURRENCE OF L-ISOASPARTYL METHYLTRANSFERASE
ACTIVITY IN THE SOLUBLE FRACTION OF PLANTS
"'LIh11t,d~ 'd~e activity (pmollminlmg of protein) Species Plant Material '-' _ L-isoAsp peptide S~
Green al~a ,~ . - C. reinhardtii .~.y~ldli.~. Gells 0.15iO.00 0.43+0.01 - Jt corn fresh kernels 0.30+0.04 1.46~0.11 dry kernels 0.71+0.03 6.86+0.42 wheat embryos (germ) 0.33+0.05 14.0+0.14 kernels 0.39+0.01 4.36+0.09 Dicots alfalfa seedlings 0.35iO.01 0.47+0.02 seeds 0.34+0.03 3.42+0.25 carrots roots 0.96+0.07 2.64~0.28 seeds 0.44+0.03 1.37+0.04 lettuce leaves 0.27+0.00 0.29+0.01 seeds 0.14+0.01 0.66+0.01 pea fresh seeds 0.24+0.04 1.31 +0.05 dry seeds 0.12+0.00 1.79+0.10 potato roots 0.19+0.01 1.04+0.00 soybean seeds 0.12+0.00 0.69+0.03 spinach leaves 0.22i0.01 1.10i0.03 seeds 2.16iO.12 2.60i0.05 tomato fruit 2.90iO.16 3.03i0.17 seeds 1.17i0.02 8.07i0.15 M lhyldliOII assays were prlN"",ed in triplicate.
EXPERIMENT 5: PURIFICATION OF METHYLTRANSFAREASE FROM WHEAT GERM
Pl. of Wheat Germ Cytosol for Enzyme F. i~i, t Raw wheat germ (150 9) was ~ dcl! in 750 mL of buffer (20 mM sodium borate (pH 9.3) 5 mM EDTA 2.4 mM 2 lll~,La~ulu~ll,a,,ol and 25 mM NaCI) and stirred for 30 min at 4C. The slurry was then squeezed through four layers of chLeseLlulll and the resulting crude ho",oyenate (585 mL) was c~"llil~,yad at 7000xy for 60 min at 4C to remove l"~",L,a"e and cell debris. The supernatant (520 mL) was poured through two layers of Miracloth to filter the floating lipid layer.
F~,.iri _:- of L-l5oaa~rJllyl~ lLl~ r ,~ae from Wheat Germ The present ~uli~h d0UII strategy was based on the partial uuliriLdliùn of the protein carboXyl Ill~lLyllld,,si~,dse reported by Trivedi et al. (Trivedi et al.(1982) Eur.J.Biochem. 128 349 354). Referring to Fig. 12, crude wheat germ cytosol (515 mL, 30 mg of proteinlmL) was lûaded onto a DE 52 (Whatman) CA 0220326~ 1997-04-21 column (9 cm diameter x 13 cm resin height, 827 mL) which was ~.u.;vusl~ equilibrated at 4C with buffer (20 mM sodium borate (pH 9.3~, 5 mM EDTA, 2.4 mM 2-mercap -t' 1, and 25 mM NaCI). T~ u: -fractions were collected at an average flow rate of 8-10 mLlmin. The loaded column was washed isocratically with 1 L of buffer followed by a 6-L gradient of 25-200 mM NaCI in the above buffer. The - 5 protein profile and the NaCI ~radient were I,d by r ~~ i ' ' ~ at 280 nm and r~ y, respectively, in the corresponding fractions. Every fifth fraction was assayed for L-;soda3,ua~
I"tlh~ laae using VYP-lL-isoAsp)-HA (SEQ ID NO:15) as the peptide substrate. One peak of methyltransferase activity was pooled ~fractions 80-110,600 mL, see brickets) and further purified by reverse - -- sulfate gradient s-' ' " as described by King (E ' ~y 11, 367-371, 1972).
The pH of the DE-52 pooled material was adjusted to 8.38 with Z0 mL of 1 M Tris-HCI, pH 7.97. 15.62 g of Celite 545 (Baker Analyzed Reagent, 11 9 of Celitel1 9 of protein) was then added with stirring to 80% aai dliull (56.1 9 of ammonium sulfatellOO mL initial volume) in a 30-min period at room temperature, and then stirring was continued for an additional 45 min. This Celite mixture cGr,i ~, JildlLd cytosolic proteins was poured into a 3 cm diameter x 19 cm column and packed with the aid of a peristalic pump at room t~ ldlL.~. The column was washed ;a~CIdi -'ly with 150 mL
(a~,.u~i",alul~ two column vol) of 80% saturated . sulfate solution c v 0.05 M Tris-HCI
(pH 7.97) and eluted with a 550 mL linear gradient d~ ~a~;"g from 80 to 0% saturation in ammonium sulfate. The flow rate of the gradient was a~J~/.uAillldluly 0.6 mLlmin, and 7.5-min fractions were collected.
The percent of a - sulfate and protein in the Gul I L -r ~ ~ fractions was d~ d by con~u.. l.. ua and dbaG~lJdllt.e at 280 nm, l~a~ ,Ii.ul`r. Every second fraction was assayed for L-;aoaspa, Irl "~lI,yll,a"a~,aae using VYP-(L-isoAsp)-HA (SErl ID Nr?:15) as the peptide substrate. Fractions (65-74), containing the highest specific activity of the m~lhylllana~ldser were pooled (95 mL, see brackets) and ~ ~l-s~ue,,ll~ purified on a Sephacryl S-200 (Sigma) gel filtration column (2 cm diameter x 77.5 cm resin high, 243 mL). Buffer co"l v 20 mM Tris-acetate (pH 7.0), 0.2 mM EDTA, 15 mM 2 mercaptoethanol, and 10 mM NaCI was used to e~. ' b,dle and run the column at 4C. The flow rate of the column was -d at 0.12 mLlmin and 30-min fractions were collected. Every second fraction was assayed for L-isoaspd, lyl ",~II,yll,ans~erdse using VYP-(L isoAsp)-HA as the peptide substrate. AbsG.L,a"ce at 280 nm was measured to d~l~, - the protein conc~lllld: of these fractions. Purified wheat germ L ;aoaa,ual Iyl u~lhyll~a".3~e~ase cona;al~"ll~ eluted in one or two fractions, roughly cu,,~,uonding to a fraction volume of 134-139 mL ~see arrow Fig. 12C).
Sul~JHa;llyly, the L-;aoaalJallyl Ill~lhyllldlla~eldse eluted in a highly purified state in a fraction nearly cu"~3l ou li 9 to the total volume of the column. This success in obtaining a highly purified enzyme pl~pdldliun from the Sephacryl S-200 gel filtration column was attributed to this unusual aLsrj",liùn n"",~
The overall ,u-l,iri~dliun of the L-;aOaS~Ial Iyl Ill~lhylllallal~ld5e from wheat germ is summarized in Table 4, and the typical pcl~e~ e C~ G~;liull cû~.3pondi,~g to each step in the purification is shown CA 0220326~ 1997-04-21 W O96/12797 PCTrUS9~/13691 in Fig. 13. Referring to Fig. 13, active fractions containing ~ hyltla~ ld~ from each purification step were analyzed by SDS-PAGE using the buffer system described by Laemmli ILaemmli (1970) A~at~re 227, 600685). Protein fractions were mixed in a ratio of 2:1 (vlv~ with sample buffer 1180 mM Tris-HCI (pH
6.8), 6.0% lwlv) SDS, 2.1 M 2-ll,~" , i ' I, 35.5% (vlv) glycerol, and 0.004% (wlv) b ~ 1l ' ' bluel and boiled for 3 min. These fractions were electrophoresed in a 12.5%(wlv) acrylamidelO.43% (wlv) N,N
"~lh~ Lnide separating gel. Gels were stained in Cc~ ~~ brilliant blue. The ' ' mass alandal~ (Bio Rad) included, ' ~ la~e b (97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21.5 kDa), and Iysozyme (14 kDa). The samples analyzed were crude wheat germ I ~ (lane A), filtered crude cytosol (lane B), fractions 80 100 from the DEAEcellulose column (lane C), reactions 65-74 from the reverse ammonium sulfate gradient ''-'- liun step (lane D), and fraction 39 from the Sephacryl S 200 column (lane E). The position of the :h~llldll~,d~e p l~ liuh~ (MT) is indicated at the right with an arrow (Fig. 13).

PURIFICATION nF L-ISOASPARTYL
METHYLTRANSFERASE FROM WHEAT GERM CYTOSOL

total total volume proteinactivity % specific sample (mL) (mg) 1, 'I. ) recovery activity ~Juliri~alh)i (u,,,oll Img) crude hc .oge,~dl~ 585 19012 69205 100 3.64 1.0 crude cytosol (7000 5Z0 15600 57616 83.3 3.69 1.0 9) DEAE cellulose 600 600 52020 75.2 86.7 23.8 reverse, - 45.3 24.9 10140 14.7 436.4 119.9 sulfate gradient solubilization Sephacryl S 200 15.0 1.04 5025 7.3 4855.1 1333 1\1 ~d at pH 6.0 using 500 ,uM VYP-(isoAsp)-HA as a methyl acceptor.
Mono Q Anion L- ' _ C! I i _ . r Fractions Golll g L-;~oa~,ud,lil m~lllyllla,,~'dse from several Sephacryl S 200 gel filtration columns were pooled and then dialyzed (Spectropor, cutoff 3500 Da) in buffer A. Dialyzed m~lllyllld"~ dse (0.6 mg of protein) was Dal,liulldl~d on a Mono Q HR 515 anion exchange (rl,d""a";d) column (5 mm diameter x 50 mm resin height, 1 mL) pl~viously ~ "S~dled with buffer A. One-minute fractions were collected at a flow rate of 0.5 mLlmin. The loaded column was washed ;~o~ldlil,ull~ with buffer A for 15 min followed by a linear gradient of O to 100% buffer B (20 mM Tris acetate ~pH 7.0), CA 0220326~ 1997-04-21 W O96/12797 PCT~US9~113691 0.2 mM EDTA, 15 mM 2-m~ a~ltL- ' 11, 10% qlycerol, and 1 M sodium acetate) over 60 min. The column effluent was ~ ~d at 280 nm. Typically m~ ltlal.~tu.a~e activity was detected in fractions - 43-44. Fractions containing active :h~ .a~e were pooled and used for ~ ~ ~' u ~' studies.
The calculated molecular mass of the major polypeptide determined by SDS p~',à~,~lamide slab gel El,,~ , h~ was 28,000 Da. It was '~ ~,dl~d that this FH~ pt ' L ll, ' to the L-;~ùas~.d. Iyl m~lh~ d..~ c~e by renaturin9 individual gel slices in the presence of Triton X-100 as described by Clarke (Clarke (1981) ~- ~ r~ ph~.Acta 670, 195-202). The purity of this, u"~. was rL~Ii",dl~d to be 86% from densitometry of the Coomassie-stained qel. The remaining minor polypeptide e( I : could be removed by an additional -' I ~ step. Dialyzed b~ LIaae was loaded onto a Mono Q anion exchan~qe column and eluted with a linear gradient of 0-1 M sodium acetate.
Active :'lylllall~teràse eluted at ~, o.~i,llalul~ 0.5 M sodium acetate.
Gh l l of L-lsoaspartyl Metl,~ ' u~e from Wheat Germ '~ :h~11lld~steldse purified through the Mono ~ step [12,500 pmollminlmg at pH 7.5 with the VYP
(isoAsp)-HA peptide as the substrate] was used to study the :.r~,;R.,;Iy of the wheat germ enzyme. Like the E. coli and human e,yll,,l~c~ hyl~l........ ;,t~ , the wheat germ enzyme ~tti.. it.,ll~ m~ ldl~s L-;~Oaa~-al Iyl residues in synthetic peptides.
EXPERIMENT 6: AMINO ACID SEQUENCE OF PURIFIED WHEAT GERM METHYLTRANSFERASE
Reverse-Phase HPLC
HG~ erL Ill~lh~lllall;~r~la~e suitable for sequence analysis was obtained by reverse-phase HPLC ~ p.,.tl.ll''a''~e liquid chromat~ , hl~) of the enzyme purified through the Sephacryl S-200 step.
Fractions were loaded onto a Vydac C-4 column (1 cm i.d. x 25 cm, 300 A pore, 5 ,um spherical silicâ
support) equilibrated with 65% solvent B and eluted with a linear gradient of 65 80% solvent B over 45 min at 3.0 mLlmin flow rate, where solvent A is 0.1% IH~lu,ùac~liL acid in water (wlv) and soivent B is 0.1% IHtlu~,,uact:li.. acid in 99% 1' ~I,'D.9% water (wlvlv). The column effluent was Illouil~,~d at 280 nm as 1-min fractions were collected. Volatile reagents were removed from the fractions in a Savant Speedvac a~,r.,.dlu~ and then these fractions were subjected to SDS-PAGE and silver staining (Jones (1990) in Current Protocols in Molecular Biology Suppl. 11, John Wiley and Sons, New York). The wheat germ lu~lllyllldll ,~lase eluted at about 40 min as a single pulr~,,.li-l~ band with an apparent molecular mass of 28,000 Da.
- 30 Amino Acid S~, D~ . by Tryptic and Si ,' ~'- aureus V8 Protease Mapping Il n;, ~ byll~_ C~ld~e suitable for amino acid sequence analysis was obtained byreverse-phase HPLC analysis as described above. This material was digested with trypsin and Slap/~/~coccu~ aureus V8 protease, and the resulting peptides were recovered by reverse-phase HPLC using a Vydac C-18 column. N-terminal Edman s~ ;.9 was then p~tu~ ed on these peptides. The partial peptide sequence data obtained were used to generate cliJL~ l~4uli.1~ probes and to confirm the presence of poly,,,u,uh;;.,,,s andlor multiple genes (see EXPERIMENT 7 below).

CA 0220326~ 1997-04-21 W O96/12797 PCTrUS95/13691 EXPERIMENT 7: cDNA CODING FOR WHEAT METHYLTRANSFERASE
Synthetic Oli~ ' t ' Probes "v- ' Jt'' probes were ;,~ ' using ,~ 5yl N,N-''' r~ phG:l~uh~
chemistry in a Gene Assembler Plus DNA synthesizer (Pha,l"d";d LKB Biotechnolo~qy). An oligollubli..~
,~u,t~ .,ti,.g the T7 promoter of the pn~ ,l SK+, ~ ~ ' I, T7 [DMT-TAATACGACTCACTATAGGG]
(SEQ ID N0:11), and three degenerateoligonucleotides, MB1 [TCTGG(GIA)AT(GIA)TG(CIT)TC(GIA)ATNCCCATl ~SEQ ID N0:12), MB3 containing an EcoRI linker [CTCGAATTCTA(CIT)lGlT)T-NAA(GIA)CA(GIA)TA(CIT)GGNGT] (SEQ ID N0:13), and MB4 containing a Hinolll linkerlTCAAAGC I I I I (GIA)TC(TIGIA)ATNAC(CIT)TGNAG] (SEn ID N0:14), were a~lllh-~;~r~d for use as probes and as primers in PCR n, "9~ of a wheat cDNA library (described below). The primers were purified by size exclusion chromatography using Bio-Spin 6 columns (Bio-Rad).
Isolation of o cDNA Clone for L-D , lyl ~' hyl~ ' ue.e From Wheat Deye~ldleS'JC' I ~lides were synthesized on the basis of the partial amino acid sequence data ~see above) and then used to amplify a region of the I Ll,~ ,ase cDNA from a wheat cDNA library co"~ d with poly (A)+ RNA isolated from 48-h-etiolated wheat seedlings (Hatfield, et al.(1990) J~7;nlt'hDm 265, 15813 15817). An 850 bp PCR product was amplified using a 384-fold desG.. .al~
ide, MB3, l~u,t~"li"y the nucleic acid sequence at the 5'-region of the ",~lh1ll,a"~,dse cDNA
Ic..ll, e ding to the peptide YLKQYGV) and a primer encoding the T7 promoter of the pBluescript vector.
The identity of the 850-bp PCR product was verified by Southern h1L~idi~dliù~ using a 64-fold d~y~:"e,dle Z ~ ' lide, MB1, IL~ul~rlllbly the nucleic acid sequence in the middle region of the meth~ll,d"~r~,d,e cDNA (r,G"~pr l'~, to the peptide GIEHIPE). In order to obtain a PCR product CGI~i ~, the hrlll~.,.;,l~dse cDNA without the pBluescript vector s, . the 850-bp PCR product was used as a template in a PCR reaction with the MB3 primer and a 288fold degel1~,dl~ e''Jc~ ' ^tide, MB4, I~,UI~b .~lilld the nucleic acid sequence at the 3'-region of the Ill~lh~lllldr,.r~,dse cDNA tcr,l,~u, " g to the peptide LQVIDK). PCR - , '"il,dlion produced a 600 bp fragment cr,r,i ' 9 only the Ill~lhyllrdn;~ dse cDNA seq~r ~ce as d~ by PCR dideoxy chain l~, ' liùn se~ g Screening of the wheat cDNA
library with this 600-bp product resulted in the isolation of one positive plaque which was rescued to a pBluescript phagemid in E. coliXL-1 pUC19 to give the pMBM1 plasmid, which was then used to lldll:~101111 E. col~ DH5a cells.
DNA Se, ~e of the Gene Encoding L-l,Oa"~ yl ~' ' yll,.. G~ se from Wheat The DNA sequence of the 952 bp cDNA insert in the plasmid pMBM1 was determined using the seu~lrll~ .9 strategy shown in Fig. 14. Referring to Fig. 14, both strands of the pMBM1 clone cu"i 3 the wheat ",rlh~lllJ,.~r~,dse cDNA insert (a 952-bp EcoRI fragment) were seqlJenced by dideoxy chain-termination seq. ~ [, . ,9 01i~nu~l~uliles were s~"ll,e ,;~ed using the sequence of a PCR product cor,l ' ' g 600 bp of the wheat ",~II,yllld"~,dse cDNA and then used as primers to sequence the 952-bp fragment as shown in the 5e4u~ll1,;1l9 strategy in this figure. The DNA sequence of the methyllld"~l~ldse cDNA and CA 0220326~ 1997-04-21 W O96/12797 PCTrUS95113691 its deduced amino acid sequence are indicated as SE~ ID NOs:5 and 6, ~ e~.li.ely. Referring to SE~ ID
NOs:5 and 6, the sequence of the coding strand of the 944-bp insert of the plasmid pMBM1 is shown without the terminal EcoRI linkers (GGAATTCC) that were added to the cDNA library. The 690-bp I"clh~lll 'c,ase cDNA initiates at the ATG codon at position 118 and lc, : - at the TGA codon at position 808. The calculated molecular weight of the 230 amino acid polypeptide deduced for the 690-bp open reading frame is 24,710. In contrast,purified ~h11LI. '~.dsemigrated as a 28,000-Da pcl~ucuL '~
as determined by SDS-PAGE (Fi~. 13).
EXPERIMENT 8: COMPARISON OF SEQUENCED PEPTIDE FRAGMENTS AND PREDICTED SEQUENCE
Comparison of S~r, ' Peptide rr_" of L~ yl ' ~ Cl .,..~ from Wheat Germ and Its ~ ' Amino Acid S, e from pMBM1 ~I lcl-c;~ d;~cl~r at 12 sites between the predicted amino acid sequence of the wheat cDNA and the sequence of the peptide fragments of the wheat germ L~ Ga~,Ual IYI Ill~lh~l~rd~ se were found (Fig. 15). Referring to Fig. 15, following digestion with trypsin (T) and S. aureL/s V8 protease (V), peptides were Ic.,u~..,bd by reverse-phase HPLC and seuut ,~Pd by dullJIlldlcnl Edman deyladaliuil. Peptide s~ s of ~I.. 9.. ~ u.. h .~d in the order of elution are shown by lines in cull~ua~i~ù~ to the deduced cDNA sequr~re The presence of a space indicates that ' ~ -L id~,"liriL.aliull of the amino acid residue could not be made in this cycle. Additional residues identified in a particular cycle are indicated in pdlcl,ll,dses above. Residues above in brackets denote an amino acid ,ul, ~liLulion at this position where no evidence was found for the cDNA-encoded residue. As a whole, the amino acid sequence of purified wheat enzyme is cun~;.lc,ed to be indicated in SEQ ID N0:7 with the following e~L~u6u"s. At position 41, A was found in T8 and V13; N was found in T9. At position 52, I was fond in T8, V14, and V17.
At position 54, only L was found in V17. At position 156, A was found in T6 and V8; V was found in T7 and V9. In six of these positions, the b,.~,~.illlclli 'l~ dcucll d amino acid sequence data clearly show the presence of an amino acid not encoded by the cDNA. At the other six positions, residues in addition to the encoded residue were identified by Edman s~lu~ g These results are COIl,;alclll with the hexaploid nature of this species of wheat, where the three diploid genomes (AABBDD) can contain alleles with variant s~, --s, leading to the I . ' of variant gene products ~Peumans, et al.(1982) Plan~
154, 562 567; Wright, et al.(1989) J.Mol.Evol. 28, 327-336). Most of the amino acid changes are iocated outside of the three highly conse,.,cd regions shared among ,llelll~ d,,~Pc~ses. It is interesting to speculate that these amino acid dirrcll `LS can result in enzymes with slightly different methyl acceptor ~uel,ifi~;lics, which would give the cell the ability to recognize and polc"lidll~ repair a wider range of damaged proteins. According to the present uuU6~aliun method, these enzyme variants can be purified as well.
EXPERIMENT 9: SKIN TREATMENT USING METHYLTRANSFERASE
Purified u,clll~llldllsferase (either isolated from plant sources or as a iccollllJ;lldlll human enzyme produced in bacteria) and S-adenosyl-L-",ci' - are mixed to final conc6''l'dliûns of 0.01% and CA 0220326~ 1997-04-21 W O96/12797 PCTrUS9~/13691 0.00004% r~s,u6c~ 'y in a water-based cream -~ i ~ glycerin and mineral oil. A skin or hair cream is ~v, ' Ird using water, glycerin, cetearyl alcohol, palm oil glyceride, Ceteareth-20, mineral oil, petrolatum, sorbitol, avocado oil, DMS0 (or othe! carrier molecules), steric acid, alantoin, squalane, lh,l~Jalab~,," Sodium Carbomer 941, r '~ : human L-;..oa~.d,lyl;D-aspartyl ,,,~lhyll~a,,~v,dve (or plant methyltransferase), propylparaben, S-adenosyl-L-methionine, Quaternium-15, fragrance, FD&C Yellow - No.5, and FD&C Red No.4. A skin mist (n~ h~' ") is ~ using water, nVlycerin, cetearyl alcohol, DMSO (or other carrier molecules), citric acid, recombinant human L ;~GaV,Ual lyliD aspartyl melhyll,d,,v~vv, d~e (or plant ",~lh~ll,a" ,~r,ase), S adenosyl-L 1' e, Qualrl 15, and r, ~, . A l, ' Illdl skin patch is formulated using the same ingredients as the above skin mist. These ~ ua~vliu~ can be directly applied to the skin. After cleaning the skin with soap and water, the ",~II,ylll ~d,dse skin cream (a), mist (b) and patch (c) are used by the following methods lcv,uvvlNel~. (a) applying a dab (0.5ml) of a viscous formulation to a central spot and spreading over an area of about 100 cm2 by rubbing with srnall circular motions directly by hand with or without latex gloves andlor with or without ar, 'il such as towels and tissues; (b) using a less viscous fvlnlvlalivn ("MethylMist") and spraying on with a spray bottle or atomizer in which the residue can be wiped into the skin as described above for the skin cream; and (c) using the same ~cllllulaliull as "MethylMist" but enclosing in a patch that adheres to skin and delivers the suL~Ia"ce through micro pores in the patch directly to the skin.
EXPERIMENT 10: OTHER MEDICAL TREATMENT USING METHYLTRANSFERASE
An injectable or topical p,~ua,dliùn (pH--7.3) for Ll~dll"~lll of the eye (e.g.,for pre~r,li"g cataracts). Injectable ~-~r~-a~dLivns for the brain (e.g., for pl~vrll01lg plaque formation) or the blood stream (e.g., for I ~ flexibility) contains 137 mM NaCI, 2.7 mM KCI, 4.3 mM Na2HP04*7H20, 1.4 mM
KH2P04, 25 mM 1. ' - I human L isoaspartyllD-aspartyl ",~II,yll~dl,s~e,dse Isdlv,àtiv..) or 1-3 ~M to match S-adenosyl-L-",~ couc~,,l,dliuns, 30 ,uM S adenosyl ",~ ' e, and 0.22 ,uM sterile filtered.
For timerelease delivery systems, the injectable preparation can be packaged in carrier 'i,-sor,es or u,uGruus ~IIULIUI~5. These plr~Jdldliùns can be directly injected to the eye, brain or blood stream.
EXPERIMENT 11: DIAGNOSIS USING METHYLTRANSFERASE
A sample of tr~L~us,u;~al fluid (0.01 ml) diluted in 0.2 M sodium citrate buffer, pH 6.0 is mixed with 0.03 ml of a mixture of S adenosyl [14C-methyl]-L ",~ (100 cpmlpmol) and purified "~rll~yllldrl~r~dse (from plant sources or human l~cvll,L;,Idnt) in the same buffer. The latter mixture contains 400 pmol of Idd~vlàLvllvd s-av~llGs~ n ,e and 10 lldcluyldl,,s of purified ",~II,ylild"a~,ase.
The mixture is incubated for 15 min at 37 C and then 0.04 ml of a solution of 0.1% sodium dodecyl sulfate, 0.2 M sodium hydroxide is added. This mixture is then applied to a piece of thick filter paper and is ~u~l,E~rlrd in a plastic vial cGni J 7 ml of liquid sci"lillaliun cocktail. After 2 hours at room 1ulll,u~ldlulr, the paper is removed and the vial counted. The ~ddiua,,6vi~y is ,u,upu,liur,al to the amount of damaged L isoaspartyl and D aspartyl residues in the cerebrospinal fluid.

CA 0220326~ 1997-04-21 W O96/12797 PCT~US95/13691 SEQUENCE LISTING
.

~1) GENERAL INFORMATION
(i) APPLICANT: THE REGENTS OF THE UNlv~sITy OF CALIFORNIA
(ii) TITLE OF THE lNV~NllON: PRODUCTION AND USE OF HUMAN AND P~ANT
METHYLTRANSFERASES
(iii) NUMBER OF SEQUENCES: 19 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Knobbe, Martens, Olson and Bear (B) STREET: 620 Newport Center Drive 16th Floor - (C) CITY: Newport Beach (D) STATE: CA
(E) COUNTRY: USA
(F) ZIP: 92660 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Diskette (B) COMPUTER: IBM Compatible (C) OPERATING SYSTEM: DOS
(D) SOFTWARE: FastSEQ Version 1.5 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Altman, Daniel E
(B) REGISTRATION NUMBER: 34,115 (C) REFERENCE/DOCKET NUMBER: UCLA010.001A
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 714-760-0404 (B) TELEFAX: 714-760-9502 (C) TELEX:
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 681 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single - (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
- (iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

CA 0220326~ 1997-04-21 W O96/12797 PCTAUS9~/13691 GCTCCACACA TGCATGCATA TGcGcTAGAA CTTCTATTTGATCAGTTGCA TGAAGGAGCT 240 AATAATGTCA GGAAGGACGA TCCAACACTT CTGTCTTCAGGGAGAGTACA G~~ GTG 420 (2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 684 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single ( D ) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 226 amino acids (B) TYPE: amino acids (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:
(ix) FEATURE:
(A) NAME/KEY: Other (B) LOCATION: 22...22 (D) OTHER INFORMATION: Ile or Leu (A) NAME/KEY:Other (B) LOCATION: 119...119 (D) OTHER INFORMATION: Ile or Val (A) NAME/KEY:Other (B) LOCATION: 205...205 CA 0220326~ l997-04-2l W O96/12797 PCT/US9~/13691 (D) OTHER INFORMATION: Lys or Arg (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Ala Trp Lys Ser Gly Gly Ala Ser His Ser Glu Leu Ile His Asn Leu Arg Lys Asn Gly Ile Xaa Lys Thr Asp Lys Val Phe Glu Val Met Leu Ala Thr Asp Arg Ser His Tyr Ala Lys Cys Asn Pro Tyr Met Asp Ser . Pro Gln Ser Ile Gly Phe Gln Ala Thr Ile Ser Ala Pro His Met His Ala Tyr Ala Leu Glu Leu Leu Phe Asp Gln Leu His Glu Gly Ala Lys Ala Leu Asp Val Gly Ser Gly Ser Gly Ile Leu Thr Ala Cys Phe Ala Arg Met Val Gly Cys Thr Gly Lys Val Ile Gly Ile Asp His Ile Lys Glu Leu Val Asp Asp Ser Xaa Asn Asn Val Arg Lys Asp Asp Pro Thr Leu Leu Ser Ser Gly Arg Val Gln Leu Val Val Gly Asp Gly Arg Met Gly Tyr Ala Glu Glu Ala Pro Tyr Asp Ala Ile His Val Gly Ala Ala Ala Pro Val Val Pro Gln Ala Leu Ile Asp Gln Leu Lys Pro Gly Gly Arg Leu Ile Leu Pro Val Gly Pro Ala Gly Gly Asn Gln Met Leu Glu Gln Tyr Asp Lys Leu Gln Asp Gly Ser Ile Lys Met Xaa Pro Leu Met Gly Val Ile Tyr Val Pro Leu Thr Asp Lys Glu Lys Gln Trp Ser Arg Trp Lys (2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUBNCE CHARACTERISTICS:
(A) LENGTH: 227 amino acids (B) TYPE: amino acids (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:
(ix) FEATURE:
(A) NAME/KEY: Other (B) LOCATION: 22...22 (D) OTHER INFORMATION: Ile or Leu (A) NAME/KEY:Other (B) LOCATION: 119...119 (D) OTHER INFORMATION: Ile or Val (A) NAME/KEY:Other (B) LOCATION: 205...205 (D) OTHER INFORMATION: Lys or Arg (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

CA 0220326~ 1997-04-21 W O96/12797 PCT~US95/13691 Ala Trp ~ys Ser Gly Gly Ala Ser His Ser Glu Leu Ile His Asn Leu 1 5 10 15 rg Lys Asn Gly Ile Xaa Lys Thr ASp Lys Val Phe Glu Val Met Leu Ala Thr Asp Arg Ser His Tyr Ala Lys Cys Asn Pro Tyr Met Asp Ser Pro Gln Ser Ile Gly Phe Gln Ala Thr Ile Ser Ala Pro His Met His Ala Tyr Ala Leu Glu Leu Leu Phe ASp Gln Leu His Glu Gly Ala Lys 80 la Leu Asp Val Gly Ser Gly Ser Gly Ile Leu Thr Ala Cys Phe Ala 95 rg Met Val Gly Cys Thr Gly Lys Val Ile Gly Ile Asp His Ile Lys Glu Leu Val ASp ASp Ser Xaa Asn Asn Val Arg Lys ASp Asp Pro Thr Leu Leu Ser Ser Gly Arg Val Gln Leu Val Val Gly Asp Gly Arg Met Gly Tyr Ala Glu Glu Ala Pro Tyr Asp Ala Ile His Val Gly Ala Ala 145 150 155 160 la Pro Val Val Pro Gln Ala Leu Ile Asp Gln heu Lys Pro Gly Gly 165 170 175 rg Leu Ile Leu Pro Val Gly Pro Ala Gly Gly Asn Gln Met Leu Glu Gln Tyr ASp Lys Leu Gln Asp Gly Ser Ile Lys Met Xaa Pro Leu Met Gly Val Ile Tyr Val Pro Leu Thr Asp Lys Glu Lys Gln Trp Ser Arg Asp Glu Leu (2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 944 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

CA 0220326~ l997-04-2l W O 96/12797 PCTrUS9~/13691 (2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 230 amino acids (B) TYPE: amino acids (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide . (iii) HYPOTHETICAL: NO
- (iv) ANTISENSE: NO
(v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Met Ala Gln Phe Trp Ala Glu Gly Ser Leu Glu Lys Asn Asn Ala Leu l 5 10 15 Val Glu Tyr Leu Lys Gln Tyr Gly Val Val Arg Thr Asp Lys Val Ala Glu Val Met Glu Thr Ile Asp Arg Ala Leu Phe Val Pro Glu Gly Phe Thr Pro Tyr Thr Asp Ser Pro Met Pro Ile Gly Tyr Asn Ala Thr Ile Ser Ala Pro His Met His Ala Thr Cys Leu Glu ~eu Leu Lys Asp Tyr Leu Gln Pro Gly Met His Ala Leu Asp Val Gly Ser Gly Ser Gly Tyr Leu Thr Ala Cys Phe Ala Met Met Val Gly Pro Glu Gly Arg Ala Val Gly Ile Glu His Ile Pro Glu Leu Val Val Ala Ser Thr Glu Asn Val Glu Arg Ser Ala Ala Ala Ala Leu Met Lys Asp Gly Ser Leu Ser Phe His Val Ser Asp Gly Arg Leu Gly Trp Pro Asp Ala Ala Pro Tyr Asp Ala Ile His Val Gly Ala Ala Ala Pro Glu Ile Pro Arg Pro Leu Leu Glu Gln Leu Lys Pro Gly Gly Arg Met Val Ile Pro Val Gly Thr Tyr Ser Gln Asp Leu Gln Val Ile Asp Lys Ser Ala Asp Gly Ser Thr Ser Val Arg Asn Asp Ala Ser Val Arg Tyr Val Pro Leu Thr Ser Arg Ser Ala Gln Leu Gln Asp Ser (2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 231 amino acids (B) TYPE amino acids (C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:
(ix) FEATURE:
(A) NAME/KEY: Other (B) LOCATION: 18...18 CA 0220326~ 1997-04-21 PCT~US9~/13691 (D) OTHER INFORMATION: Asp or Glu (A) NAME/KEY:Other (B) LOCATION: 41...41 (D) OTHER INFORMATION: Asn or Ala (A) NAME/KEY:Other (B) LOCATION:52...52 (D) OTHER INFORMATION: Ile or Thr (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Met Ala Gln Phe Trp Ala Glu Gly Ser Leu Glu Lys Asn Asn Ala Leu Val Xaa Tyr Leu Lys Gln Tyr Gly Val Val Arg Thr Asp Lys Val Ala Glu Val Met Glu Thr Ile Asp Arg Xaa Leu Phe Val Pro Glu Gly Phe Thr Pro Tyr Xaa Asp Xaa Pro Met Pro Ile Gly Tyr Asn Ala Thr Ile Ser Ala Pro His Met His Ala Thr Cys Leu Glu Leu Leu Lys Asp Tyr Leu Gln Pro Gly Met His Ala Leu Asp Val Gly Ser Gly Ser Gly Tyr Leu Thr Ala Cys Phe Ala Met Met Val Gly Pro Glu Gly Arg Ala Val Gly Ile Glu His Ile Pro Glu Leu Val Xaa Ala Ser Thr Glu Asn Val GlU Arg Ser Ala Ala Ala Ala Leu Met Lys Asp Gly Ser Leu Xaa Phe His Val Xaa Asp Gly Arg Leu Gly Trp Pro Asp Xaa Ala Pro Tyr Asp Ala Ile His Val Gly Ala Ala Ala Pro Glu Ile Pro Arg Pro Leu heu Glu Gln Leu Lys Pro Gly Gly Arg Met Val Ile Pro Val Gly Thr Tyr Ser Gln Asp Leu Gln Val Ile Asp Lys Ser Xaa Asp Gly Ser Thr Xaa Val Xaa Asn Asp Ala Xaa Val Arg Tyr Val Pro Leu Thr Ser Arg Ser Ala Gln Leu Gln Asp Ser (2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 68 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

(2) INFORMATION FOR SEQ ID NO:9:

CA 0220326~ 1997-04-21 W O96/12797 PCT~US95/13691 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1218 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOhOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
' ' (vi) ORIGINAL SOURCE:
(ix) FEATURE:
(A) NAME/KEY: Coding Sequence (B) LOCATION: 85...765 (D) OTHER INFORMATION:

(xi) SEQu~N~ DESCRIPTION: SEQ ID NO:9:
GTAATACGAC TCACTATAGG GCGAATTGGG TACCTCGAGT CTAGAGGATC W"l''l'~'l''l"l'AA 60 Ala Trp Lys Ser Gly Gly Ala Ser His l 5 Ser Glu Leu Ile His Asn Leu Arg Lys Asn Gly Ile Ile Lys Thr Asp Lys Val Phe Glu Val Met Leu Ala Thr Asp Arg Ser His Tyr Ala Lys Cys Asn Pro Tyr Met Asp Ser Pro Gln Ser Ile Gly Phe Gln Ala Thr Ile Ser Ala Pro His Met His Ala Tyr Ala Leu Glu Leu Leu Phe Asp CAG TTG CAT GAA GGA GCT A~A GCT CTT GAT GTA GGA TCT GGA AGT GGA 351 Gln Leu His Glu Gly Ala Lys Ala Leu Asp Val Gly Ser Gly Ser Gly ATC CTT ACT GCA TGT TTT GCA CGT ATG GTT GGA TGT ACT GGA A~A GTC 399 .Ile Leu Thr Ala Cys Phe Ala Arg Met Val Gly Cys Thr Gly Lys Val Ile Gly Ile Asp His Ile Lys Glu Leu Val Asp Asp Ser Val Asn Asn Val Arg Lys Asp Asp Pro Thr Leu Leu Ser Ser Gly Arg Val Gln Leu Val Val Gly Asp Gly Arg Met Gly Tyr Ala Glu Glu Ala Pro Tyr Asp 140 145 ' 150 Ala Ile His Val Gly Ala Ala Ala Pro Val Val Pro Gln Ala Leu Ile . . . . . . . . . _ _ _ _ _ _ CA 0220326~ 1997-04-21 W O96/12797 PCT/US9~/13691 . -36-Asp Gln Leu Lys Pro Gly Gly Arg Leu Ile Leu Pro Val Gly Pro Ala Gly Gly Asn Gln Met Leu Glu Gln Tyr Asp Lys Leu Gln Asp Gly Ser Ile Lys Met Lys Pro Leu Met Gly Val Ile Tyr Val Pro Leu Thr Asp Lys G~u Lys Gln Trp Ser Arg Asp Glu Leu CACAGCTTGT TTTGGACTTT GTTACACTGT TATTTTCAGC ATGAAAATGT GT~lllllll 909 TTGGACACTT AATTCTGTTCTGATATTAATTTATCAGATTG~llll~lGCATTGGATAAC 1149 (2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 324 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

(2) INFORMATION FOR SEQ ID NO~
(i) SEQUENCE CHARACTERISTICS:
(A) L~NGTH: 20 base pairs (B) TYP~: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:

.. .. ... . .. . _ . _ . .. . . . . .. . . . . . .. .

W 096/127~7 PCT~US95/13691 (2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) hENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOhECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
- (iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DBSCRIPTION: SEQ ID NO:12:

(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOl~llCAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

(2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids (B) TYPE: amino acids (C) STRANDEDNESS: single (D) TOPOLOGY: linear PCTIUS9~/13691 (ii) MO~ECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:
(ix) FEATURE:
(A) NAME/KEY: Modified Base (B) LOCATION: 4...4 (D) OTEER INFORMATION: L-isoaspartyl (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
Val Tyr Pro Xaa His Ala (2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUBNCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acids (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:
(ix) FEATURE:
(A) NAME/KEY: Modified Base (B) LOCATION: 5...5 (D) OTHFR INFORMATION: L-isoaspartyl (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
Lys Ala Ser Ala Xaa Leu Ala Lys Tyr (2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids (B) TYPE: amino acids (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:
(ix) FEATURE:
(A) NAME/KEY: Modified Base (B) LOCATION: 3...3 (D) OTHER INFORMATION: L-isoaspartyl (A) NAME/KEY:Modi~ied Base (B) LOCATION: 4...4 (D) OTHER INFORMATION: Phe-NH2 PCTrUS9~/13691 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Ala Ala Xaa Xaa (2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids (B) TYPE: amino acids (C) STRANDEDNESS: single . (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide ~iii) HYPOTHETICAL. NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:
(ix) FEATURE:
(A) NAME~KEY: Modi~ied Base (B) LOCATION: 4...4 (D) OTHER INFORMATION: D-aspartyl (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
Val Tyr Gly Xaa Pro Ala (2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE ~HARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acids (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:
(ix) FEATURE:
(A) NAME/KEY: Modified Base (B) LOCATION: 5...5 (D) OTHER INFORMATION: D-aspartyl (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l9:
Lys Ala Ser Ala Xaa Leu Ala Lys Tyr

Claims (62)

WHAT WE CLAIMED IS:

1. An isolated recombinant human L-isoaspartyl/D-aspartyl protein methyltransferase obtained by expression of a polynucleotide having a sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:2.

2. An isolated recombinant human L-isoaspartyl/D-aspartyl protein methyltransferase having an amino acid sequence selected from the group consisting of SEQ NO:3 and SEQ ID NO:4.

3. An isolated polynucleotide having the coding sequence, of the sequence indicated as SEQ
ID NO:5, said polynucleotide coding for a plant L-isoaspartyl protein methyltransferase.

4. An isolated recombinant plant L-isoaspartyl protein methyltransferase obtained by expression of SEQ ID NO:5.

5. A purified plant L-isoaspartyl protein methyltransferase having the amino acid sequence SEQ ID NO;7, obtained from wheat germ.

6. A method of recombinantly producing human L-isoaspartyl/D-aspartyl protein methyltransferase, comprising:
modifying a plasmid that contains the T7 promoter region and the full coding region of human L-isoaspartyl/D-aspartyl protein methyltransferase, using oligonucleotides, to provide multiple cloning sites, an efficient ribosome binding site, and a strong translational initiator region, said initiator region being designed to function in bacterial and/or eukaryotic expression system;
transfecting the constructed vector into a host that contains an inducible T7 polymerase gene;
and inducing overexpression of the methyltransferase, whereby the methyltransferase is produced.

7. The method of Claim 6, wherein the inducible T7 polymerase gene is induced by heat shock or inclusion of a chamical and the inducing step comprises addition of IPTG.

8. The method of Claim 7, wherein the said chamical inducing the T7 polymerase gene is isopropyl .beta.-D-thiogalactopyranoside (IPTG)

9. The method of recombinantly producing human L-isoaspartyl/D-aspartyl protein methyltransferase according to Claim 6, wherein said full coding region is that of isozyme II of human L-isoaspartyl/D-aspartyl protein methyltransferase having the sequence indicated as SEQ ID NO:2.

10. The method of recombinantly producing human L-isoaspartyl/D-aspartyl protein methyltransferase according to Claim 9, wherein said plasmid to be modified is obtained from a commercial cDNA library derived from human brain tissue using a radiolabeled 769 bp HaeIII fragment from the coding region of a 1580 bp murine methyltransferase cDNA.

11. The method of recombinantly producing human L-isoaspartyl/D-aspartyl proteinmethyltransferase according to Claim 10, wherein the plasmid to be modified is plasmid pDM2 having the sequence indicated as SEQ ID NO:9.

12. The method of recombinantly producing human L-isoaspartyl/D-aspartyl proteinmethyltransferase according to Claim 6, wherein said modification is conducted by replacing the region between the T7-promoter site and the start codon of the enzyme, which is 107 bp from the Kpnl site to the Narl site, with a synthetic fragment containing ribosome binding and initiator sites, said fragment having the sequence indicated as SEQ ID NO:8.

13. The method of recombinantly producing human L-isoaspartyl/D-aspartyl proteinmethyltransferase according to Claim 6, wherein said host is E. coli strain BL21(DE3) or any other suitable strain.

14. The method of recombinantly producing human L-isoaspartyl/D-aspartyl proteinmethyltransferase according to Claim 6, wherein the concentration of said IPTG is 0.03 mM to 8 mM.

15. A method of purifying recombinantly produced human L-isoaspartyl/D-aspartyl protein methyltransferase present in a lysed bacterial extract in which methyltransferase expression has occurred, comprising:
adding a nucleotide precipitant to the extract to remove DNA present in the extract subsequent to removing the cellular debris therefrom;
precipitating the methyltransferase with ammonium sulfate;
removing the ammonium sulfate by dialysis; and purifying the methyltransferase from the dialysate by using a DEAE-cellulose anion-exchange chromatography column.

16. The method of purifying recombinantly produced human L-isoaspartyl/D-aspartyl protein methyltransferase according to Claim 15, wherein said nucleotide precipitant is protamine sulfate or polyethyleneimine.

17. The method of purifying recombinantly produced human L-isoaspartyl/D-aspartyl protein methyltransferase according to Claim 15, wherein the addition of said protamine sulfate is 0.1 volumes of a 4% solution thereof.

18. The method of purifying recombinantly produced human L-isoaspartyl/D-aspartyl protein methyltransferase according to Claim 15, wherein the saturation of said ammonium sulfate is 50% to 60%.

19. The method of purifying recombinantly produced human L-isoaspartyl/D-aspartyl protein methyltransferase according to Claim 15, wherein said column is fully equilibrated before the loading of the dialysate.

20. A method of purifying plant L-isoaspartyl protein methyltransferase from wheat, comprising:
obtaining a crude cytosol of raw wheat;
fractionating the crude cytosol by DEAE-cellulose chromatography;
adding ammonium sulfate to the pooled active fractions in the presence of a protein carrier;
fractionating the resulting material by reverse ammonium sulfate gradient solubilization; and purifying the pooled active fractions by gel filtration chromatography, whereby the methyltransferase is purified as a monomeric 28,000 Da species.

21. The method of purifying plant L-isoaspartyl protein methyltransferase according to Claim 20, wherein said crude cytosol is obtained from wheat germ.

22. The method of purifying plant L-isoaspartyl protein methyltransferase according to Claim 20, wherein said fractionation by the DEAE-cellulose chromatography is conducted at pH 9.3.

23. The method of purifying plant L-isoaspartyl protein methyltransferase according to Claim 20, wherein said sulfate is added to 80% saturation.

24. The method of purifying plant L-isoaspartyl protein methyltransferase according to Claim 20, wherein said protein carrier is Celite 545.

25. The method of purifying plant L-isoaspartyl protein methyltransferase according to Claim 20, wherein said reverse ammonium sulfate gradient solubilization is conducted with a linear gradient decreasing from 80% to 0% saturation in ammonium sulfate.

26. The method of purifying plant L-isoaspartyl protein methyltransferase according to Claim 20, wherein said pooled active fractions after the fractionation by reverse ammonium sulfate gradient solubilization contains 26-31% saturated a ammonium sulfate.

27. The method of purifying plant L-isoaspartyl protein methyltransferase according to Claim 20, wherein said gel filtration chromatography is Sephacryl S-200 gel filtration chromatography.

28. The method of purifying plant L-isoaspartyl protein methyltransferase according to Claim 20, further comprising, subsequent to the gel filtration chromatography, dialyzing the purified methyltransferase and introducing the dialyzed methyltransferase to a Mono Q anion exchange column.

29. An expression vector for human L-isoaspartyl/D-aspartyl protein methyltransferase constructed by modifying a plasmid that contains the full coding region of the human L-isoaspartyl/D-aspartyl protein methyltransferase, using oligonucleotides, to provide multiple cloning sites, an efficient ribosome binding site, and a strong translational initiator region, said initiator region being designed to function in a bacterial and/or eukaryotic expression system.

30. The expression vector according to Claim 29, wherein said full coding region is that of isozyme II of human L-isoaspartyl/D-aspartyl protein methyltransferase having the sequence indicated as SEQ
ID NO:2.

31. The expression vector according to Claim 30, wherein the plasmid to be modified is plasmid pDM2.

32. The expression vector according to Claim 29, wherein said modification is conducted by replacing the region between the T7-promoter site and the start codon of the enzyme, which is 107 bp from the Kpnl site to the Narl site, with a synthetic fragment containing ribosome binding and initiator sites, said fragment having the sequence indicated as SEQ ID NO:8.

33. A method of treatment for a medical condition associated with an increase inL-isoaspartyl/D-aspartyl residues of polypeptides in a tissue, comprising:
administering to the tissue an amount of methyltransferase sufficient to convert said L-isoaspartyl/D-aspartyl residues to L-aspartyl residues in the tissue.

34 The method of treatment for a condition according to Claim 33, wherein said condition is crosslinking of matrix proteins and degradation of flexibility of tissues.

35. The method of treatment for a condition according to Claim 33, wherein said condition is cataracts.

36. The method of treatment for a condition according to Claim 33, wherein said condition is degradation of corneal flexibility.

37. The method of treatment for a condition according to Claim 33, wherein said condition is formation of plaque in brain tissues.

38. The method of treatment for a condition according to Claim 33, wherein said condition is degradation of cellular function in brain tissues.

39. The method of treatment for a condition according to Claim 33, wherein said condition is degradation of flexibility in a vascular system.

40. The method of treatment for a condition according to Claim 33, wherein said condition is infertility related to eggs and/or sperm.

41. The method of treatment for a condition according to Claim 33, wherein said condition is formation of fibrosis in tissues.

42. The method of treatment for a condition according to Claims 33, wherein saidmethyltransferase is an isolated recombinant human L-isoaspartyl/D-aspartyl protein methyltransferase

43. The method of treatment for a condition according to Claim 33, wherein said methyltransferase is administered in conjunction with S-adenosylmethionine.

44. The method of treatment for a condition according to Claim 33, wherein said methyltransferase is a purified plant L-isoaspartyl protein methyltransferase.

45. A method of diagnosis of a state where L-isoaspartyl/D-aspartyl residues are accumulated in a patient, comprising:
obtaining a biological sample containing protein from said patient; and measuring, the content of L-isoaspartyl/D-aspartyl residues accumulated in said protein, by using methyltransferase as a probe.

46. A pharmaceutical preparation for treatment of a condition associated with an increase in L-isoaspartyl/D-aspartyl residues of polypeptides in a tissue, comprising:
human L-isoaspartyl/D-aspartyl protein methyltransferase, and a pharmaceutically acceptable carrier.

47. The pharmaceutical preparation for treatment of a condition according to Claim 46, wherein said preparation is a skin or hair cream.

48. The pharmaceutical preparation for treatment of a condition according to Claim 46, wherein said preparation is eye drops.

49. The pharmaceutical preparation for treatment of a condition according to Claim 46, wherein said preparation is an injectable solution.

50. The pharmaceutical preparation for treatment of a condition according to Claim 46, wherein said preparation contains S-adenosylmethionine as a substrate of the enzyme.

51. A method of treatment for plant protein degradation associated with an increase in L-isoaspartyl residues of polypeptides in a tissue, comprising:
administering to the tissue an amount of plant L-isoaspartyl protein methyltransferase sufficient to convert said L-isoaspartyl residues to L-aspartyl residues in the tissue.

52. The method of treatment for plant protein degradation according to Claim 51, wherein said plant protein degradation is caused by desiccation, aging, and environmental stress.

53. The method of treatment for plant protein degradation according to Claim 51, wherein said treatment is applied to seed tissues and/or seedling tissues.

54. The method of treatment for plant protein degradation according to Claim 51, wherein said methyltransferase is administered in conjunction with S-adenosylmethionine.55. A method of recombinantly producing plant L-isoaspartyl protein methyltransferase, comprising:
modifying a plasmid that contains the T7 promoter region and the full coding region of plant

L-isoaspartyl protein methyltransferase, using oligonucleotides, to provide multiple cloning sites, an efficient ribosome binding site, and a strong translational initiator region, said initiator region being designed to function in bacterial and/or eukaryotic expression system;
transfecting the constructed vector into a host that contains an inducible T7 polymerase gene;
and inducing overexpression of the methyltransferase, whereby the methyltransferase is produced.

56. A method of purifying recombinantly produced plant L-isoaspartyl protein methyltransferase present in a lysed bacterial extract in which methyltransferase expression has occurred, comprising:
adding a nucleotide precipitant to the extract to remove DNA present in the extract subsequent to removing the cellular debris therefrom;
precipitating the methyltransferase with ammonium sulfate;
removing the ammonium sulfate by dialysis; and purifying the methyltransferase from the dialysate by using a DEAE-cellulose anion-exchange chromatography column.

57. The method of Claim 56, wherein the nucleotide precipitant comprises protamine sulfate or polyethyleneimine.

58. A methyltransferase for use as a medicament in the treatment of a medical condition associated with an increase in L-isoaspartyl/D-aspartyl residues of polypeptides in a tissue.

59. The methyltransferase of Claim 58, wherein, said condition is crosslinking of matrix proteins and degradation of flexibility of tissues.

60. The methyltransferase of Claim 58, wherein said condition is cataracts, degradation of corneal flexibility, formation of plaque in brain tissues, degradation of cellular function in brain tissues, degradation of flexibility in a vascular system, infertility related to eggs and/or sperm, or formation of fibrosis in tissues.

61. The methyltransferase of Claim 58, wherein said methyltransferase is an isolated recombinant human L-isoaspartyl/D-aspartyl protein methyltransferase.

62. The methyltransferase of Claim 58, in combination with S-adenosylmethionine.63. The methyltransferase of Claim 58, wherein said methyltransferase is a purified plant L-isoaspartyl protein methyltransferase.