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WO1998008865A1 - DOMAINE DE FIXATION DE COIFFE DU FACTEUR D'INITIATION eIF-4E DE SYNTHESE PROTEIQUE EUCARYOTE HUMAINE - Google Patents

DOMAINE DE FIXATION DE COIFFE DU FACTEUR D'INITIATION eIF-4E DE SYNTHESE PROTEIQUE EUCARYOTE HUMAINE Download PDF

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Publication number
WO1998008865A1
WO1998008865A1 PCT/US1997/015295 US9715295W WO9808865A1 WO 1998008865 A1 WO1998008865 A1 WO 1998008865A1 US 9715295 W US9715295 W US 9715295W WO 9808865 A1 WO9808865 A1 WO 9808865A1
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Prior art keywords
peptide
leu
arg
cap
mrna
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PCT/US1997/015295
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English (en)
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WO1998008865A9 (fr
Inventor
Dixie J. Goss
Diana Friedland
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Life Technologies, Inc.
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Priority to EP97942384A priority Critical patent/EP0975652A1/fr
Priority to AU44094/97A priority patent/AU4409497A/en
Publication of WO1998008865A1 publication Critical patent/WO1998008865A1/fr
Publication of WO1998008865A9 publication Critical patent/WO1998008865A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity

Definitions

  • the present invention is in the field of molecular biology Specifically, the invention provides truncated peptides of the eukaryotic protein synthesis initiation factor eIF-4E comprising the region of eIF-4E that binds to the cap regions of mRNA molecules The invention also provides methods and kits using these peptides suitable for use in the isolation of capped mRNA molecules and in the production of full-length cDNA molecules
  • the initiation phase of eukaryotic protein synthesis is characterized by recognition of the m 7 (5 ')Gppp(5 ')N (where "N” is any nucleotide) cap structure at the 5' terminus of eukaryotic mRNA by initiation factors (elF's) (reviewed in Rhoads, R.E , Trends Biochem. Sci. (Pers. Ed.) 13:52-56 (1988); Sonenberg, N., Prog. Nucleic Acid Res. Mol. Biol.
  • cap recognition by eEF-4E is a limiting step in protein synthesis is supported by the observation that it is the least abundant of all the initiation factors and thus may serve a regulatory function (Duncan, R.F., et al., J. Biol.. Chem. 262:380-388 (1987); Hire ath, L.S., et al., J. Biol.. Chem. 260:7843-7849 (1985)).
  • this protein is a key point for post-transcriptional control of gene expression.
  • EGF and other growth regulatory peptides rapidly stimulate the phosphorylation of eIF-4E phosphorylation in intact cells (Donaldson, R.W., et al., J. Biol.. Chem. 266:3162-3166 (1991); Sonenberg, N manipulate Biochemie 76:839-846 (1994); Rhoads, R E., J. Biol. Chem. 268:3011-3020 (1993); Proud, C.G., Curr. Top. Cell. Regul. 32:243-369 (1992)).
  • the amino acid sequence for the cap-binding protein has been previously reported for several species including yeast (Altmann, M., et ⁇ /., M?/. Cell. Biol.. 7:998-1003 (1987)), wheat (Metz, A M , et al, Nucl. Acids Res. 20:4096 (1992)), human (Rychlik, W., et al., Proc. Natl. Acad. Sci. U.S.A. 5- ⁇ :945-949 (1987)), and mouse (Alt ann, M, et al., . Biol. Chem.
  • the cap-binding subunit of wheat germ eIF-4F lacks any such acidic residue in these regions, containing instead a ⁇ -turn-inducing proline residue in the region of tryptophan 5 and a basic histidine residue in the region of tryptophan 7 (Metz, A M , et al., Nucleic Acids Res. 20:4096 (1992))
  • Immobilized aluminum(III)-chelate chromatography has facilitated the isolation of binding site peptides due to the presence of highly charged phosphates on the photoinserted azidonucleotide which interact with the Al 3+ (Salvucci, M.E., et al, Biochemistry 57:4479-4487 (1992); Shoemaker, M.T., and Haley, B E., Biochemistry 52:1883-1890 (1993); Jayaram, B., and Haley, B E., J. Biol. Chem. 269:3233-3242 (1994); Anderssen, L. J., Chromatography 539: 327-334 (1991)).
  • Isolated cap-binding eIF-4E proteins have several uses.
  • U.S. Patent No. 5,219,989 to Sonenberg et al. discloses the preparation of multifunctional fusion proteins comprising the eIF-4E protein and at least a second protein having the ability to bind to a solid support matrix.
  • the exemplified second protein is protein A which is capable of binding to a resin having an IgG antibody attached to it.
  • Such fusion proteins may be used for the preparation of a cDNA library mostly containing full-length cDNA and for the purification of capped mRNA.
  • Such a preparation would provide a less expensive and more specific means for binding of capped mRNA molecules, and for production of full-length cDNA molecules, than the fusion proteins of U.S. Patent No. 5,219,989.
  • such a preparation could be produced more efficiently in recombinant systems such as mammalian host cells, since the nucleic acid sequences directing the synthesis of such a preparation would be smaller and therefore more efficiently incorporated and transcribed than those for fusion proteins.
  • the present invention provides a truncated peptide of the eIF-4E protein consisting essentially of the mRNA cap-binding domain of eIF-4E, wherein the peptide has an amino acid sequence corresponding to that set forth in SEQ ED NO' 1.
  • the invention further encompasses variant peptides comprising the cap-binding domain of eIF-4E within a ⁇ -sheet structural motif
  • the invention provides the peptides immobilized on a solid support, a resin comprising the peptides immobilized on a solid support, a method for isolating a capped mRNA molecule, a method for producing a full-length cDNA molecule, a kit for isolating a capped mRNA molecule and a kit for producing a full-length cDNA molecule
  • Other preferred embodiments of the present invention will be apparent to one of ordinary skill in light of the following drawings and description of the invention, and of the claims
  • FIG. 1A is a graph and an autoradiogram of a gel demonstrating saturation of [ ⁇ - 32 P]8-
  • FIG. IB is a graph and an autoradiogram of a gel demonstrating competitive Inhibition of [ ⁇ - 32 P]8- ⁇ 3 GTP photoincorporation into r eIF-4E by m 7 GTP.
  • r eIF-4E (4 micrograms) was incubated with 40 ⁇ M [ ⁇ - 32 P]8-N 3 GTP in the presence of increasing concentrations of m 7 GTP under the conditions described for Fig 1A 32 P incorporation was determined by radioisotopic imaging and quantitation
  • FIG. 2 is a bar graph representing a chromatogram (radioactivity profile) of immobilized
  • FIG. 3A is a chromatogram of microbore, C 8 reverse phase HPLC of tryptic peptide fractions from Al 3 ' chelate chromatography, demonstrating a typical UV profile of the radioactive fractions resulting from K 2 HPO 4 elution of the Al 3 ⁇ chelate column
  • FIG. 3B is a chromatogram demonstrating the corresponding 32 P cpm profile of the fractions resulting from the HPLC in Fig. 3A Radioactivity levels were determined by liquid scintillation counting.
  • Binding studies of m 7 GTP, m 7 GpppG, and various capped mRNAs with eIF-4E have provided insight into the nature of the eIF-4E*»cap interaction (Carberry, S E., et al, Biochemistry 25:8078-8083 (1989); Carberry, S.E., et al., Biochemistry 57:1427-1432 (1992), Goss, D.J., et al., Biochem. Biophys. Ada.
  • the photoaffinity probe [ ⁇ - 32 P]8- N 3 GTP was used as a photoactivable analogue of the m 7 G cap structure of mRNA to selectively label the m 7 GTP cap-binding domain of human recombinant eIF-4E ( r eEF-4E)
  • the photolabeled peptide resulting from proteolysis was isolated using a!uminum(III)-chelate chromatography followed by reversed-phase high performance liquid chromatography
  • the amino acid sequence of this peptide has been identified in the present invention as consisting essentially of residues 113- 122 of the amino acid sequence of human eIF-4E described previously (Rychlik, W., et al., Proc. Natl. Acad Sci. U.S.A. 5 ⁇ :945-949 (1987))
  • This peptide thus contains tryptophan 6 (Trp 113 in full-length eIF-4E)
  • the present invention is thus related to a truncated cap binding protein (e.g eIF-4E protein) which is capable of binding to the 5' terminus cap structure of mRNA produced in eukaryotic cells
  • truncated proteins comprise at least amino acids Trp 113 to Arg I22 corresponding to the human eIF-4E sequence as disclosed by Rychlik, W.. et al, Proc. Natl. Acad. Sci. U.S.A. 5* ⁇ :945-949 (1987), or suitable variants thereof
  • truncated proteins include
  • Variants of the truncated eIF-4E protein include any change or changes (deletions, substitutions or additions) in or to the amino acid sequence, provided that the variant retains the ability to bind to the cap region of mRNA.
  • the ability to retain cap binding function can be determined by the assays described herein Preferably, changes are made so as to conserve the hydrophobicity and charge of the peptide. Examples of such variants include but are not limited to one or more substitutions within the peptide as follows
  • ⁇ -strands which are organized into higher secondary structures such as ⁇ -sheets and ⁇ / ⁇ barrels in a variety of proteins, share certain general properties with respect to their preferred amino acid sequences (Sternberg, M J E , et al, Phil. Trans. R. Soc. Lond B 293 177- 189 ( 1981 ), Wodak, S J , et al. , Biochem. Soc. Symp. 57 99- 121 ( 1990), Siezen, R J , et al., Prot. Eng. 4(7) 719-737 (1991), Chothia, C , et al., Ciba Found.
  • ⁇ -strands comprising ⁇ -sheet structural motifs in most proteins are composed of stretches of nonpolar or hydrophobic amino acid residues and are typically 3-10 residues in length While any hydrophobic residue may be found in these regions, including alanine (Ala), valine (Val), leucine (Leu), isoleucine (He), phenylalanine (Phe), methionine (Met), tryptophan (Trp), proline (Pro) or cysteine (Cys), the branched aliphatic residues (Leu, He and Val) are particularly preferred (Stemberg, M J E , et al, Phil.
  • ⁇ -sheet motifs typically have one or more charged (especially aspartic acid (Asp) or glutamic acid (Glu)), aromatic (especially tyrosine (Tyr) or threonine (Thr)) or ⁇ -turn-inducing (especially Pro or glycine (Gly)) residues immediately prior to and/or following the stretch of hydrophobic amino acids (Stemberg, M.J.E., et al, Phil. Trans. R Soc. Lond B 293:177-189 (1981); Wodak, S.J., etal, Biochem. Soc. Symp.
  • the present invention also encompasses variants of the eIF-4E peptide described above (for SEQ ID NO: l) in which Trpl 13 (tryptophan 6) is maintained in the same ⁇ -sheet structural motif. Included in these variants are peptides having an amino acid sequence corresponding to the general pattern
  • XI is a charged, aromatic or ⁇ -turn-inducing amino acid, most preferably Asp,
  • X2 is a hydrophobic amino acid, most preferably Leu, He, Val or Trp, and the subscripts for XI and X2 indicate the number of such residues in consecutive sequence at the indicated position.
  • X is any amino acid
  • n is a whole number ranging in value from 0 to 70, preferably 0 to 50, 0 to 25 or 0 to 10.
  • peptides of the invention include those having an amino acid sequence corresponding to the general pattern X n -T ⁇ -Lys-Arg-Gly-Gly-Arg-Trp-Leu-Ile-Thr-Leu-Asn-Lys-Gln-Gln-Arg-X n (SEQ ID NO 4)
  • X is any amino acid
  • n is a whole number ranging in value from 0 to 70, preferably 0 to 50, 0 to 25 or 0 to 10.
  • X is any amino acid
  • n is a whole number ranging in value from 0 to 70, preferably 0 to 50, 0 to 25 or 0 to 10.
  • Other preferred variant peptides of the invention include those having an amino acid sequence corresponding to the general pattern
  • XI is generally a charged, aromatic or ⁇ -turn-inducing amino acid (such as Asp, Glu, Tyr, Thr, Gly or Pro)
  • X2 is a hydrophobic amino acid (such as Leu, He, Val or Trp) and the subscripts for XI and X2 indicate the number of such residues in consecutive sequence at the indicated position.
  • variant peptides include, but are not limited to: T ⁇ -Lys-Arg-Gly-Gly-Arg-Trp-Leu-Ile-Thr-Leu-Asn-Lys-Gln-Gln-Arg (SEQ ID NO: 7);
  • Truncated eIF-4E peptides such as those described above may be prepared according to well-known methods of solid phase peptide synthesis, using routine methods of organic and inorganic chemistry Alternatively, the peptides may be produced by recombinant DNA techniques, which would yield a recombinant peptide comprising the above-described truncated eIF-4E peptides Production of such recombinant peptides is preferably accomplished by expression of the peptide in a host cell, including a bacterial, yeast or mammalian cell, in which the peptide coding sequence is operably linked to a promoter sequence Typically, the promotor- peptide encoding sequence is contained by a vector Techniques for isolation of nucleic acids, insertion into a vector, transformation of a host cell and isolation of the recombinant protein from the host cell are well-known in the art (see, for example, Sambrook, J , et al, eds , Molectdar Clo
  • the peptides produced as described above may be covalently or non-covalently immobilized on a solid phase support
  • solid phase support any solid support to which a peptide can be immobilized
  • Such solid phase supports include, but are not limited to nitrocellulose, diazocellulose, glass, polystyrene, polyvinylchloride, polypropylene, polyethylene, dextran, Sepharose, agar, starch, nylon, beads and microtitre plates
  • Linkage of the peptide of the invention to a solid support can be accomplished by attaching one or both ends of the peptide to the support Attachment may also be made at one or more internal sites in the peptide.
  • Attachment can be via an amino acid linkage group such as a primary amino group, a carboxyl group, or a sulfhydryl (SH) group or by chemical linkage groups such as with cyanogen bromide (CNBr) linkage through a spacer
  • an affinity tag sequence to the peptide can be used such as GST (Smith, D B and Johnson, K.S. Gene 67:31 (1988); polyhistidines (Hochuli, E et al., J. Chromatog. 411 11 (1987)); or biotin.
  • Such affinity tags may be used for the reversible attachment of the peptide to the support
  • Such immobilized peptides are useful in chromatographic isolation of mRNA molecules by binding to the cap region of the mRNAs.
  • a solution containing the capped mRNA molecules would be contacted, en masse or on a column, with the resin containing the immobilized cap-binding peptides of the present invention. After allowing binding of the mRNA molecules to the resin, unbound materials may be simply washed away.
  • the mRNA molecules may then be rapidly removed from the resin or other solid phase support by contacting the resin or support with an elution buffer which may contain, for example, a competitive analog of the mRNA cap such as m 7 GDP (see U.S. Patent No. 5,219,989), a buffer that causes a change in the pH, and/or ionic strength of the solution.
  • Immobilized capped mRNA molecules may instead be used directly in an RT-PCR method according to methods that are well known.
  • the mRNA cap-binding peptides of the present invention can be used to obtain full-length cDNAs, as has been described for eIF-4E protein A fusion proteins (U.S. Patent No. 5,219,989).
  • Production of cDNA molecules is effected by reverse transcription of a desired mRNA with a protein having reverse transcriptase activity, for example, according to U.S. Patent No. 5,244,797 with reverse transcriptase, or with a DNA polymerase having reverse transcriptase activity.
  • the reverse transcriptase is a truncated or mutated reverse transcriptase that lacks RNase H activities that gives a much higher proportion of full-length cDNA compared to the wild type reverse transcriptase.
  • Such reverse transcriptases (SuperscriptTM and Superscript II TM respectively) may be obtained from Life Technologies, Inc. (Gaithersburg, MD)
  • DNA polymerases having reverse transcriptase activity are thermostable enzymes.
  • DNA polymerases having reverse transcriptase activity include the wild type and mutant Tne enzymes (see WO96/ 10640 and copending application entitled "Cloned DNA Polymerases from Thermotoga and Mutants thereof by A. John Hughes and Deb K. Chatterjee, filed August 14, 1996); Tth enzyme (U.S. Patent Nos. 5,192,674, 5,413,926, 5,242,818 and WO91/09950); and Taq (U.S. Patent Nos. 5,474,920, 5,079,3524,889,818 and 5,352,600). See also U.S. Patent Nos.
  • RNA specific nuclease such as a T. or T 2 nuclease
  • the reverse transcriptase copies the entire length of the mRNA, or if it falls short by a few nucleotides such that there is no unhybridized GpN residue in the corresponding mRNA, T, will not degrade the mRNA and, as a result, the cap structure will remain covalently bound to the mRNA.cDNA hybrid If, however, cDNA synthesis was not complete, the single-stranded RNA specific nuclease will degrade unpaired RNA and remove the cap structure from the mRNA cDNA hybrid
  • the mRNA cDNA hybrids are incubated with the truncated eIF-4E protein of the present invention, which may be immobilized on a solid phase support as described above As a result of this incubation, only those mRNA cDNA hybrids that have a covalently attached cap structure will bind to the protein of the present invention
  • immobilizing the truncated eIF-4E to a solid phase support all of the non-capped containing hybrids, or incomplete cDNAs, will wash away
  • the bound full-length capped mRNA cDNA hybrids may then be released competitively by treatment with a cap analog such as m 7 GDP as described in U S 5,219,989
  • the resulting purified fraction contains only full length or near full length first strand cDNAs which then act as templates for second strand synthesis
  • the steps for completing the cDNA library are the same as those normally used by those of ordinary skill in the art
  • the present invention is a major advance in the art
  • the present invention is also directed to a kit for isolation of capped mRNA molecules
  • Kits according to this aspect of the invention comprise a carrier means having in close confinement therein one or more container means, each of which contain the reagents described -1*6-
  • a first container means may contain a truncated eIF-4E peptide of the present invention, which may be bound to a solid phase as described above.
  • a second container means may contain a competitive analogue of the cap region of mRNA molecules, which is preferably m 7 GDP.
  • Kits according to this aspect of the invention comprise a carrier means having in close confinement therein one or more container means, each of which contain the reagents described herein.
  • a first container means may contain the truncated eIF-4E protein of the present invention, which may be bound to a solid phase as described above.
  • a second container means may contain a nuclease, which is preferably T, nuclease.
  • a third container means may contain a protein having reverse transcriptase activity, which is preferably a truncated or mutant reverse transcriptase lacking RNAse H activity.
  • a fourth container means may contain an elution buffer which may contain a competitive analogue of the cap region of mRNA molecules, which is preferably m 7 GDP.
  • a fifth container means may contain one or more nucleoside triphosphates and at least one buffer salt. Additional container means may contain an oligo(dT) primer or a control RNA. Two or more of the enzymes described above may alternatively be combined into a single container means
  • the enzymes may be present in a buffered solution or in lyophilized form.
  • the enzymes may be stabilized by drying in the presence of a sugar such as trehalose (U.S. patent nos 5,098,893 and 4,824,938) or acacia gum, pectin, carboxymethyl cellulose, carboxymethylhydroxyethyl cellulose, guar, carboxy guar, carboxymethylhydroxypropyl guar, laminaran, chitin, alginates or carrageenan.
  • Sequencing grade modified trypsin was from Boehringer Mannheim Biochemica (Indianapolis, IN). Electrophoresis reagents were from Bio-Rad (Melville, NY).
  • HPLC reagents were from E M. Science (Gibbstown, NJ). All other reagents were obtained from
  • Recombinant eIF-4E Human recombinant eIF-4E was purified from Escherichia coli containing a T7 polymerase-driven vector which was described in detail elsewhere (Baker et al., J. Biol. Chem. 267: 1 1495-1 1499 (1992). See also Rychlik et al, Proc. Natl. Acad. Sci USA 5 ⁇ :945-949 (1987)). Recombinant eIF-4E was purified from bacterial lysates by m 7 GTP Sepharose affinity chromatography followed by Mono Q FPLC (Pharmacia, Piscataway, NJ) (Haas D W. & Hagedom C.H., Arch. Biochem. Biophys.
  • the isoelectric point of the recombinant eIF-4E was identical to that of the dephosphorylated iso-species of eEF-4E isolated from cultured human cells (Bu, X. & Hagedom, CH. FEBS Lett. 507:15-18 (1992)).
  • the recombinant eIF-4E prepared using these methods is phosphorylated in vitro by protein kinase C at the same sites as native eIF-4E (Haas D.W. & Hagedorn C.H., Second Messengers and Phosphoproteins 14:55-63 (1992)). Synthesis of Photoaffinity Probe.
  • the radioactive photo probe [ ⁇ - 32 P]8-N 3 GTP (specific activity, 32 mCi/ ⁇ mol) was synthesized and purified as previously reported (Potter, R.L. & Haley, B.EMethods Enzymol. 97:613-633 (1983)).
  • Photoaffinity Labeling of r eIF-4E To demonstrate saturation effects, samples containing 4 ⁇ g of r eIF-4E in photolysis buffer (10 mM Tris»Cl, no salt, no DTT, pH 7.6), were incubated at 4°C in 1.5 ml Eppendorf tubes with the appropriate concentration of [ ⁇ - 32 P]8- N 3 GTP for 60 seconds.
  • the reaction mixture was then irradiated for 90 s from a distance of 1 cm with a hand held 254 nm UVB UV lamp (intensity, 1400 ⁇ W/cm 2 ).
  • the total reaction volume was 50 ⁇ l.
  • the reaction was quenched by the addition of 250 ⁇ l of cold acetone.
  • the sample was kept at 4°C for 3 hours and then centrifuged in a Savant HSC10K high speed centrifuge at 10,000 ⁇ m for 20 minutes.
  • PSM protein solubilizing mixture
  • the pellet was resuspended in 2 ml of 2 M urea in 75 M NH 4 HCO 3 and the pH adjusted to 8.5-9.0 by the addition of concentrated NH 4 OH.
  • r eIF-4E was proteolyzed by the addition of modified trypsin (10% w/v), with shaking overnight at 20 C C Immobilized Aluminum(III)-Chelate Chromatography.
  • Iminodiacetic acid-epoxy- activated Sepharose (2 ml) was placed into a 15 ml centrifuge tube and washed as follows 3 x 10 ml distilled water, 4 x 10 ml 50 mM A1C1 3 , 3 x 10 ml distilled water, 3 x 10 ml buffer A (50 mM NH 4 OAC, pH 6 0), 3 x 10 ml buffer B (100 mM NH 4 OAC and 0 5 M NaCl, pH 7.0), 3 x 10 ml buffer A The resin was then transferred to a 10 ml disposable column (Bio-Rad) The labeled r eIF-4E digestion mixture was brought to a total volume of 3 ml with buffer A, the pH was adjusted to 6 0 with concentrated acetic acid, and the sample was loaded onto the column To increase the recovery of the labeled peptide the sample tube was washed with 2 ml buffer A The column was successively eluted
  • the percent maximal protection of 12% is determined by dividing the [ ⁇ 32 P]8-N 3 GTP concentration (40 uM) by the total maximum nucleotide plus probe concentration (340 uM). This result provides evidence that photoinsertion of [ ⁇ - 32 P]8-N 3 GTP was indeed occurring at the m 7 G cap binding site of eIF-4E.
  • GTP 21% of control
  • GDP 26% of control
  • ATP 36% of control
  • Capped mRNA was able to dramatically reduce photoinsertion (5% of control) as compared to equimolar concentrations of m 7 GTP (18% of control) and unmethylated nucleotides (maximum of 21% of control).
  • TCA was initially used to precipitate photolabeled r eEF- 4E, however this approach resulted in low yields of peptide for sequencing probably due to the disruption of the acid sensitive probe-peptide bond
  • acetone precipitation of photolabeled r eIF-4E resulted in higher yields of labeled peptide.
  • Photolabeled r eIF-4E was digested overnight with modified trypsin
  • the labeled peptide was isolated by affinity chromatography and further purified by reversed-phase HPLC, using the methods described above.
  • the flow through fractions fractions 1-5), washes (fractions 6-25), and phosphate elutions (fractions 26-31) (Fig 2) were separately pooled and subjected to reversed-phase HPLC
  • the flow through and wash fractions contained no radioactive peaks as determined by liquid scintillation counting thus no labeled peptides
  • these fractions did contain many peptide fragments, demonstrating that most of the peptides are unmodified and not retained on the Al 3 ' resin
  • the HPLC absorbance profile at 220 nm and the corresponding 32 P cpm profile for the phosphate elution fractions 16-31) is shown in figure 3.
  • the first peak at 3 minutes represents the flow through and injection disturbance and contained no peptides as revealed by sequencing data
  • the radioactivity detected with this peak was free photolyzed [ ⁇ - 32 P]8- ⁇ 3 GTP, or probe hydrolyzed during HPLC due to the lability of the N-glycosyl bond to acidic conditions (King, S , Kim, H , & Haley, B., Methods Enzymol. 196:449-466 (1991))
  • the second major radioactive peak at 23 minutes was concentrated for amino acid sequence analysis.
  • RNA:peptide binding of capped mRNA to the peptide was determined in gel shift assays under various ionic and buffer conditions In a binding solution of 20 mM HEPES, pH 7.6, 1 mM DTT,
  • the peptide resulting from photoinsertion of [ ⁇ - 32 P]8-N 3 GTP and subsequent isolation has been identified as the tryptic peptide consisting of residues 1 13-122 and thus contains tryptophan 6 Detection of the actual site of nitrene insertion is often difficult due to the lability of the N-glycosyl bond to the acidic conditions of HPLC (King, S., Kim, H., & Haley, B., Meth. Enzymol. 196:449-466 (1991)).
  • lysine 119 is the modified residue for two reasons: the photolabeled binding site peptide is the product of tryptic cleavage at Arg-112 and Arg-122 yet Lys-119 remained uncleaved; and amino acid sequence analysis was unable to identify Lys-119 despite the continuation of sequencing for three residues beyond this position.
  • Such a gap in sequencing usually indicates reaction of the probe with the missing amino acid (Shoemaker, M.T. & Haley, B.E., Biochemistry 32: 1883-1890 (1993)).
  • the amino acid is "missing" because it does not elute in the expected position when the probe has been covalently attached to it.
  • residues 3 may be charged, aromatic or beta- turn- inducing residues (such as D, E, Y, T, G or P) ; residue at position 2 may be a hydrophobic amino acid (such as L, I, V or W) "
  • Trp Leu lie Thr Leu Asn Lys Gin Gin Arg Gly Asp lie Val Val Leu 1 5 10 15
  • Trp Leu lie Thr Leu Asn Lys Gin Gin Arg Asp Gly Val Val lie lie 1 5 10 15 val Leu Leu Asp Gly 20
  • Trp Leu lie Thr Leu Asn Lys Gin Gin Arg Asp Leu Leu Val lie Val 1 5 10 15
  • Trp Leu lie Thr Leu Asn Lys Gin Gin Arg Glu Asp Val lie Val Leu 1 5 10 15

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Abstract

La fixation du facteur d'initiation à la structure de coiffe 5'm7G d'eucaryotes de l'ARN messager (ARN-m) signale le démarrage de la synthèse protéique. Selon la présente invention, le domaine de fixation de coiffe au site de fixation du facteur d'initiation eIF-4E a été identifié comme la région contenant la séquence allant de Trp?113 à Arg122¿. C'est pourquoi, dans un premier mode de réalisation préféré, la présente invention concerne un peptide tronqué de la protéine de fixation de coiffe comportant essentiellement le domaine de fixation de coiffe de l'ARN-m. D'une manière spécifique, l'invention concerne le peptide de fixation de coiffe eIF-4E et ses variantes. Dans d'autres modes de réalisation préférés, l'invention porte sur une molécule isolée d'acide nucléique codant pour un peptide tronqué de la protéine eIF-4E comportant essentiellement le domaine de fixation de coiffe de eIF-4E de l'ARN-m. L'invention se rapporte également à un vecteur recombiné et à une cellule hôte comprenant la molécule d'acide nucléique décrite ci-dessus et un peptide recombiné. L'invention concerne en outre les peptides tronqués ou recombinés fixés sur un support solide, à savoir une résine comprenant les peptides tronqués ou recombinés fixés sur un support solide, des procédés et des outils d'isolement de la molécule coiffée d'ARN-m et des procédés et des outils de production d'une molécule d'ARN-c complète.
PCT/US1997/015295 1996-08-30 1997-09-02 DOMAINE DE FIXATION DE COIFFE DU FACTEUR D'INITIATION eIF-4E DE SYNTHESE PROTEIQUE EUCARYOTE HUMAINE WO1998008865A1 (fr)

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EP97942384A EP0975652A1 (fr) 1996-08-30 1997-09-02 DOMAINE DE FIXATION DE COIFFE DU FACTEUR D'INITIATION eIF-4E DE SYNTHESE PROTEIQUE EUCARYOTE HUMAINE
AU44094/97A AU4409497A (en) 1996-08-30 1997-09-02 Cap-binding domain of human eukaryotic protein synthesis initiation factor eif-4e and the use thereof

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US2488196P 1996-08-30 1996-08-30
US60/024,881 1996-08-30
US92087497A 1997-08-29 1997-08-29
US08/920,874 1997-08-29

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999021985A1 (fr) * 1997-10-28 1999-05-06 Shanghai Second Medical University Cbfbtb01: facteur humain d'initiation de traduction de type tif4e
WO2001004289A1 (fr) * 1999-07-13 2001-01-18 Incyte Pharmaceuticals, Inc. PROCEDES ET COMPOSITIONS PERMETTANT DE PRODUIRE DES BANQUES D'ADNc PLEINE LONGUEUR
US6436676B1 (en) 1999-07-13 2002-08-20 Incyte Genomics, Inc. Methods and compositions for producing full length cDNA libraries
EP1326881A4 (fr) * 2000-09-19 2004-08-11 Univ Emory Preparation de arnm a coiffe
US7074556B2 (en) 1999-03-02 2006-07-11 Invitrogen Corporation cDNA synthesis improvements

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
J. BIOCHEM., 1994, Vol. 116, No. 3, MORINO et al., "Direct Expression of a Synthetic Gene in Escherichia Coli: Purification and Physicochemical Properties of Human Initiation Factor 4E", pages 687-693. *
PROC. NATL. ACAD. SCI. U.S.A., February 1987, Vol. 84, No. 4, RYCHLIK et al., "Amino Acid Sequence of the mRNA Cap-Binding Protein from Human Tissues", pages 945-949. *
PROTEIN SCI., January 1997, Vol. 6, No. 1, FRIEDLAND et al., "Identification of the Cap Binding Domain of Human Recombinant Eukaryotic Protein Synthesis Initiation Factor 4E Using a Photoaffinity Analogue", pages 125-131. *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999021985A1 (fr) * 1997-10-28 1999-05-06 Shanghai Second Medical University Cbfbtb01: facteur humain d'initiation de traduction de type tif4e
US7074556B2 (en) 1999-03-02 2006-07-11 Invitrogen Corporation cDNA synthesis improvements
WO2001004289A1 (fr) * 1999-07-13 2001-01-18 Incyte Pharmaceuticals, Inc. PROCEDES ET COMPOSITIONS PERMETTANT DE PRODUIRE DES BANQUES D'ADNc PLEINE LONGUEUR
US6369199B2 (en) 1999-07-13 2002-04-09 Incyte Genomics, Inc. Fusion protein comprising an eIF-4E domain and an eIF-4G domain joined by a linker domain
US6436676B1 (en) 1999-07-13 2002-08-20 Incyte Genomics, Inc. Methods and compositions for producing full length cDNA libraries
US6703239B2 (en) 1999-07-13 2004-03-09 Incyte Corporation Nucleic acid encoding a fusion protein comprising an EIF-4E domain and an EIF-4G domain joined by a linker domain
EP1326881A4 (fr) * 2000-09-19 2004-08-11 Univ Emory Preparation de arnm a coiffe
US6841363B2 (en) 2000-09-19 2005-01-11 Emory University Preparation of capped mRNA

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