WO2002095013A2 - Engineered bira for in vitro biotinylation - Google Patents

Abstract

An engineered biotin ligase (BirA) includes a chitin binding domain tag and a polyhistidine tag. The engineered BirA may be used to biotinylate proteins such as staphylokinase in a protein purification scheme.

Description

ENGINEERED BirA FOR IN VITRO BIOTINYLATION

FIELD OF THE INVENTION

The present invention relates to engineered biotin ligase polypeptides and polynucleotides. In particular, the invention relates to an engineered BirA for use in in vitro biotinylation of target biomolecules and tagging of BirA to facilitate its purification and subsequent removal from a biotmylated sample.

BACKGROUND OF THE INVENTION

A major reason for using Bacillus subtilis as an expression host for heterologous target protein production is its capability to secrete extracellular proteins into the culture medium. To take full advantage of this system, an efficient method of recovering the target protein is crucial. For secretory proteins which cannot be purified by a simple scheme, in vitro biotinylation using biotin ligase (BirA) offers an effective alternative for their purification. Availability of large amounts of quality BirA can be critical for methods involving in vitro biotinylation.

"Designer affinity purification" of target proteins (1), a strategy which involves fusing the target protein with an affinity tag to facilitate its purification, is an attractive approach for efficient purification of target proteins from a crude preparation. Many affinity tags have been developed for this purpose, among which glutathione S-transferase (GST) (2), P-galactosidase (3), maltose binding protein (4), and biotin acceptor domains (5, 6) are among the more popular. Since some of these proteins tags are relatively large, often contributing to more than 50% of the molecular mass of the protein fusions, their use can create some undesirable side effects. For example, large tags can occasionally cause solubility problems during protein purification or production, or they can adversely affect the conformation and biological activity of the target proteins (7, 8). Moreover, use of these tags usually requires post-purification tag removal by chemical or enzymatic means, which can be a challenging, time-consuming and costly process, and which may not be compatible with the target protein (1). For these reasons, small tags including His-tags (9), strep-tags (10) and biotinylation tags (11, 12) are often preferred affinity tags. Strep-tags I and II are peptides of 9 amino acids in length, that can selectively bind to streptavidin (Kd -10^"5 M for tag 1 and 10^"6 M for tag2). Biotinylation tags, which may be identified through screening of a peptide library (11, 12, 13), are peptides of 13-15 amino acids in length, that can be biotinylated errzymatically using the E. coli biotin ligase (BirA). Since the affinity of both His- and strep-tags for their respective affinity matrices is not particularly high, the presence of contaminants in purified target protein preparations is a common problem associated with using these tags. This shortcoming may be avoided by the use of biotinylation tag, as biotin binds to monomeric avidin (14, 15) or nitro-avidin (16) with higher affinity (K_d 10^"7 M for monomeric avidin). A systematic comparison of the use of these three tags to purify a rat neurotensin receptor expressed in E. coli demonstrated that the biotinylation tag provides the highest efficiency and purity (17).

Recombinant proteins with biotinylation tags are commonly biotinylated in vivo, using endogenous BirA or co-expressed BirA (6, 17, 18, 19). This approach may not be desirable for some applications, as in vivo biotinylation has a number of drawbacks. First, incomplete biotinylation of the target proteins often occurs, because cellular resources such as BirA and ATP are limited (18). Incomplete biotinylation caused by BirA deficiency can sometimes be overcome by co-expressing heterologous BirA (17), however, if it is caused by depletion of intracellular ATP, it is more difficult to remedy. In the biotinylation reaction, BirA uses the energy from ATP to covalently link the biotin moiety to the lysine residue on the biotinylation tag (20). Thus, the events involved in producing biotinylated fusion proteins in vivo, including the synthesis of BirA, the synthesis of the target protein and the enzymatic biotinylation, are energy-demanding processes. This may explain why in some cases, coexpression of BirA can only partially improve the biotinylation efficiency, leaving a large amount of the target protein unbiotinylated (17, 18). A second drawback with in vivo biotinylation is the presence of endogenous biotinylated proteins. For example E. coli expresses biotin carboxyl carrier protein or BCCP, a subunit of acetyl-CoA carboxylase (5). B. subtilis has two endogenous biotinylated proteins, BCCP and pyruvate carboxylase(21). Although these endogenous proteins represent a tiny fraction of the total intracellular protein, after affinity purification they are often present in undesirable amounts, in an otherwise highly purified sample. Thirdly and finally, a serious drawback to using in vivo biotinylation is that it will not effectively biotinylate secreted proteins, since both BirA and ATP are intracellular. Thus, in vivo biotinylation can't be used in conjunction with secretory production of heterologous proteins, the latter of which provides numerous advantages for protein expression and production, such as ease of protein recovery, reduced cell toxicity and absence of intracellular biotinylated contaminants, as mentioned earlier. It may be desirable therfore, to avoid the drawbacks mentioned above, to perform the biotinylation reaction in vitro.

For in vitro biotinylation, the availability of a pure and biologically active BirA protein preparation is a critical factor in the success of the biotinylation reaction. This is particularly true if large scale protein purification via biotinylation is the desired goal. Therefore, there is a need in the art for a method of producing large quantities of substantially pure and biologically active BirA, which can then be used for in vitro biotinylation methods.

SUMMARY OF THE INVENTION

In one aspect, the present invention relates to the engineering and production of a biotin ligase protein. In a preferred embodiment, the invention relates to the engineering and production of an E. coli BirA. hi one embodiment, the engineered BirA includes a C-terminal His-tag and an N-terminal chitin binding domain (CBD). Both tags facilitate the purification of this engineered version of BirA. i addition, the N-terminal CBD also allows the rapid removal of BirA from the biotinylation mixture after the completion of the reaction. In another aspect, the invention relates to polynucleotides which encode for polypeptides of the present invention. The invention further relates to vectors, expression systems and host cells which comprise such polynucleotides.

In another aspect, an engineered protein of the present invention may be used to biotinylate a biomolecule such as intracellular or secretory proteins in a purification method. In one embodiment, the secretory protein is a recombmant staphylokinase from Bacillus subtilis. The recombinant staphylokinase may be tagged with a biotinylation peptide and purified using affinity chromatography. This method of in vitro biotinylation with subsequent affinity purification using a monomeric avidin or nitro-avidin column may result in large amounts of active high purity staphylokinase.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 : Effects of biotin (A) and growth temperature (B) on the distribution of CBD-BirA-His in the intracellular fractions of E. coli. (A) Cultures were grown at 30°C. Lanes 1&2, no biotin in culture medium; lanes 3&4, 12 μM biotin in culture medium. (B) 12 μM biotin in all samples. Lanes 1&2, cultures grown at 30°C; lanes 3&6, cultures grown at 30°C and shifted to 25°C post IPTG induction; lanes 1&4, E. coli BL21(DE3)[pET-CBD-BirA-His]; lanes 5&6, negative control, E. coli BL21(DE3)[pET29b]. Samples were analyzed by SDS-PAGE. M, marker; S, soluble fraction; I, insoluble fraction. Arrow indicates CBD-BirA-His band.

Figure 2: Purification of E. coli CBD-BirA-His using: Ni²⁺ column (A), and chitin affinity column (B). (A) lane 1, crude lysate; lane 2: column flow-through; lane 3, eluates (60 mM imidazole); lane 4, eluate (250 mM imidazole); lane 5, eluate (?? μM imidazole). (B) Lane 1, crude lysate; lane 2, column flow-through; lane 3, pooled washes (2-column volumes); lane 4, eluate. Samples were analyzed by SDS-PAGE. M, marker. Arrow indicates CBD-BirA-His band.

Figure 3: Protein biotinylation using CBD-BirA-His purified on chitin affinity column, Coomassie blue-stained gel (A), Western blot (B). Samples were analyzed on a 12% SDS gel. M, marker. Lane 1 , MBP-AviTag substrate; lane 2, SAK with 15-mer biotinylation peptide tag, substrate. Biotinylation reaction was carried out as described in Materials and Methods.

Figure 4: pH activity profile of engineered ii. coli CBD-BirA-His. 100 ng of unbiotinylated MBP-AviTag was coated on the wells of Reacti-bind maleic anhydride-activated polystyrene strip plate (Pierce, USA) to act as the substrate. Reaction mixtures contained 10 mM ATP, 10 mM magnesium acetate, 50 μM biotin and 10 ng CBD-BirA-His purified by chitin affinity chromatography. Biotinylation reaction was carried out at 30°C for 20 min. Bound biotin was detected by streptavidin-horseradish peroxidase (Pierce) with 1-step slow TMB-ELISA (Pierce) as the color development reagent. The following buffers were used at 50 mM: glycine (2.5), NaAc (4.5), MES (5.5), BIS-TRIS (6.5), TRIS-HC1 (7.5), bicine (8.3, 9), CAPS (10, 11). Data represent the average of three independent trials.

Figure 5: Staphylokinase activity as determined by the radial caseinolysis assay. (A) Coomassie bluestained SDS gel showing SAK produced by B. subtilis. Amounts of samples loaded on the lanes were normalized to cell density. M, marker. Lane 1 , natural, untagged SAK produced by WB800[pSAKP] (33); lane 2, unbiotinylated SAK-PFB produced by WB800[pSAKPFB]; lane 3, purified biotinylated SAK-PFB produced by WB800[pSAKPFB]; lane 4, negative control WB800[pWB980]. (B) SAK activity was estimated using the top agarose plasminogen-skim milk plate method. The amount of SAK in the individual wells was identical to that in the corresponding lanes shown in (A). Picture was taken at 10 hours after incubation at 37°C. Numbers 1-4 correspond to the numbering in (A).

Figure 6: Purification of SAK-PFB from the culture supernatant of B. subtilis WB800[pSAKPFB] by in vitro biotinylation and monomeric avidin agarose chromatography. Samples were analyzed by SDS -PAGE and stained by Coomassie blue. M, marker. Lane 1, ammonium sulfate precipitate before biotinylation; lane 2, ammonium sulfate precipitate after biotinylation; lane 3, column flow-through; lane 4, 1 -column volume wash; lanes 5&6, eluate; lane 7, concentrated pure SAK-PFB. 5 Figure 7 is a restriction map of plasmid pET-CBD-BirA-His.

Figure 8 shows the nucleotide sequence of plasmid pET-CBD-BirA-His.

DESCRIPTION OF THE INVENTION

10

In one aspect, the invention relates to engineered proteins and polynucleotides, as described in greater detail below. In particular, the invention relates to engineered proteins and polynucleotides of an E. coli BirA comprising a C-terminus His-tag and an N-terminus chitin binding domain tag. The invention relates especially to a tagged 15 BirA having the amino acid and nucleotide sequences set out in Table 1 below as SEQ ID NO:2 and SEQ ID NO:l respectively.

Table 1

SEQ ID NO:l (coding sequence for CBD-BIRA-HIS)

10 20 30 40 50 60

I I I I I I

1 ATGACGACAA ATCCTGGTGT ATCCGCTTGG CAGGTCAACA CAGCTTATAC TGCGGGACAA

61 TTGGTCACAT ATAACGGCAA GACGTATAAA TGTTTGCAGC CCCACACCTC CTTGGCAGGA

121 TGGGAACCAT CCAACGTTCC TGCCTTGTGG CAGCTTCAAG ATCTGGGTAC CCTGGTGCCA

181 CGCGGTTCCA TGGCGATATC GGATCCGAAT TCTAAGGATA ACACCGTGCC ACTGAAATTG

241 ATTGCCCTGT TAGCGAACGG TGAATTTCAC TCTGGCGAGC AGTTGGGTGA AACGCTGGGA

301 ATGAGCCGGG CGGCTATTAA TAAACACATT CAGACACTGC GTGACTGGGG CGTTGATGTC

361 TTTACCGTTC CGGGTAAAGG ATACAGCCTG CCTGAGCCTA TCCAGTTACT TAATGCTAAA

421 CAGATATTGG GTCAGCTGGA TGGCGGTAGT GTAGCCGTGC TGCCAGTGAT TGACTCCACG

481 AATCAGTACC TTCTTGATCG TATCGGAGAG CTTAAATCGG GCGATGCTTG CATTGCAGAA

541 TACCAGCAGG CTGGCCGTGG TCGCCGGGGT CGGAAATGGT TTTCGCCTTT TGGCGCAAAC

601 TTATATTTGT CGATGTTCTG GCGTCTGGAA CAAGGCCCGG CGGCGGCGAT TGGTTTAAGT

661 CTGGTTATCG GTATCGTGAT GGCGGAAGTA TTACGCAAGC TGGGTGCAGA TAAAGTTCGT

721 GTTAAATGGC CTAATGACCT CTATCTGCAG GATCGCAAGC TGGCAGGCAT TCTGGTGGAG

781 CTGACTGGCA AAACTGGCGA TGCGGCGCAA ATAGTCATTG GAGCCGGGAT CAACATGGCA

841 ATGCGCCGTG TTGAAGAGAG TGTCGTTAAT CAGGGGTGGA TCACGCTGCA GGAAGCGGGG

901 ATCAATCTCG ATCGTAATAC GTTGGCGGCC ATGCTAATAC GTGAATTACG TGCTGCGTTG

961 GAACTCTTCG AACAAGAAGG ATTGGCACCT TATCTGTCGC GCTGGGAAAA GCTGGATAAT

1021 TTTATTAATC GCCCAGTGAA ACTTATCATT GGTGATAAAG AAATATTTGG CATTTCACGC

1081 GGAATAGACA AACAGGGGGC TTTATTACTT GAGCAGGATG GAATAATAAA ACCCTGGATG

1141 GGCGGTGAAA TATCCCTGCG TAGTGCAGAA AAACAAAAGC TTGCGGCCGC ACTCGAGCAC

1201 CACCACCACC ACCACTGA SEQ ID NO : 2 ( CBD-BIRA-HIS protein sequence)

10 20 30 40 50 60

I I I I I I

1 MTTNPGVSAW QVNTAYTAGQ LVTYNGKTYK CLQPHTSLAG WEPSNVPALW QLQD GTLVP 61 RGSMAISDPN SKDNTVPLK IALLANGEFH SGEQ GETLG MSRAAINKHI QTLRD GVDV 121 FTVPGKGYSL PEPIQLLNAK QILGQLDGGS VAVLPVIDST NQYLLDRIGE SGDACIAE 181 YQQAGRGRRG RK FSPFGAN LYLSMFWRLE QGPAAAIG S LVIGIVMAEV LRKLGADKVR 241 VKWPNDLYLQ DRKLAGILVE LTGKTGDAAQ IVIGAGINMA MRRVEΞSWN QGWITLQEAG 301 INLDRNTLAA MLIRΞLRAAL ELFEQEGLAP YLSRWEKLDN FINRPVKLII GDKEIFGISR 361 GIDKQGALLL EQDGIIKPWM GGEISLRSAE KQKLAAALEH HHHHH

5

Engineered Proteins

The invention includes an engineered protein consisting of or comprising of the formula:

10

CBD - L1 - X (I)

or the formula:

15 CBD - LI - X - L2 - (His)_n (II)

wherein CBD is a chitin binding domain, LI is a linker comprising a short amino acid sequence, X is a biotin ligase, L2 is a linker comprising a short amino acid sequence, and His is histidine and "n" is an integer equal to or between 4 and 12, preferably 20 equal to or between 5 and 8 and most preferably 6. The dashes represent covalent bonds.

Although the engineered protein described herein has CBD at the amino end of the protein and His at the carboxy end, the two tags may be reversed within the scope 25 of the present invention. Similarly, a protein of formula I above may have CBD at either the amino or carboxy end.

Biotin Ligase (X) Because the engineered protein includes a biotin ligase, also known as biotin holoenzyme synthetase or biotinyl protein ligase, the engineered protein is characterized by its ability to biotinylate proteins. Biotinylation activity may be determined by ELISA, as further described below, h one embodiment, the biotin ligase is the BirA protein of Escherichia coli referred to herein as "BirA" and includes biologically active variants thereof. Among the particularly preferred embodiments of the invention are variants of BirA polypeptide encoded by naturally occurring alleles of the BirA gene. One embodiment of an engineered protein of the invention which includes BirA shall be referred to herein as CBD-BirA-His.

Homologs ofE. coli BirA are known in the art and are included within the scope of biotin ligases used in the present invention. A biotin ligase for use in the present invention may also be obtained from other organisms of the same taxonomic genus, or from organisms of the same taxonomic family or order. For example, Salmonella typhi, Pseudomonas aeruginosa, Haemophilus influenzae, Bacillus subtilis, Bacillus halodurans, Streptococcus pneumonia, Clostridium perfringens, amongst others, are known to synthesize biotin ligases. BirA homologs are not highly conserved and sequence identity is relatively low. However, a single lysine residue, Lysl 83, appears to is strictly conserved between BirA homologs. This conserved lysine is located at position 253 in SEQ ID NO:2, above. Because of the poor sequence homology, BirA homologs, and other biotin ligases of the present invention, are characterized by biological activity as opposed to sequence homology.

Biological activity of the polypeptides of the present invention may be determined by ELISA (13), using unbiotinylated MBP-AviTag (Avidity, USA) as the substrate, as described in more detail below. Specific activity of the polypeptide may be defined as ng biotinylated MBPAviTag formed per min per μg of polypeptide at 30°C. Commercially available BirA (Avidity, LLC) has a specific activity of about 27, while preferred embodiments of the present invention have a specific activity of greater than 40. An engineered protein of the present invention is considered to have biological activity if it has a specific activity of at least about 5, preferably greater than 10, more preferably greater than about 20, and most preferably equal to or greater than the specific activity of commercially available BirA.

The biotin ligase of the invention includes the portion of the engineered protein shown in Table 1 identified as amino acid residues 72 to 391 [SEQ ID NO:2] as well as proteins, polypeptides and peptides with BirA activity, regardless of the degree of identity of the protein, the polypeptide, or the peptide, to BirA. A polypeptide or peptide is a sequence of amino acids that is entirely the same as part of, but not all of, any amino acid sequence of any biotin ligase of the invention.

hi addition to the standard single and triple letter representations for amino acids, the term "X" or "Xaa" may also be used in identifying certain amino acids of the engineered protein of the invention. "X" and "Xaa" mean that any of the twenty naturally occurring amino acids may appear at such a designated position in the protein sequence.

Chitin Binding Domain (CBD)

In one embodiment, the CBD sequence of the polypeptide described above is the 53 amino acid chitin binding domain of chitinase Al of Bacillus circulans (Residues 1 - 53 in SEQ ID NO:2). Within the scope of the invention, the CBD moeity may be any structure which confers an affinity for chitin to the engineered protein, including various amino acid sequences of known chitin binding domains. This affinity (minimum JCj of about 10^"6) permits the engineered protein to be purified by affinity chromatography using chitin as the ligand.

Other examples of CBD which may be incorporated into the present invention are the CBDs of chitinase from Kurthia zopfii, Serratia marcescens, Pseudomonas aeruginosa, Aeromonas sp. (strain No. 10S-24), and of other enzymes such as a putative dioxygenase from Streptomyces coelicolor.

Linker (LI) In one embodiment, the linker that connects the CBD portion of the engineered protein to the biotin ligase portion is the 18 amino acid sequence identified by residues 54 - 71 in SEQ ID NO:2.

Polyhistidine Tag ((His)_n)

In one embodiment, the polyhistidine tag is a C-terminal hexahistidine, which allows the protein to be purified by metal chelation chromatography. The polyhistidine tag may comprise 4 to 12 histidine residues, preferably 5 to 8 residues and most preferably 6 residues.

Linker (L2)

In one embodiment, the linker that connects the biotin ligase portion of the engineered protein to the polyhistidine linker is the 8 amino acid sequence identified by residues 392 - 399 in SEQ ID NO:2. The linker should not form any stable secondary structure (i.e.in random configuration) so that it would not interfere the folding of each domain (i.e. BirA and polyHis). It should also be hydrophilic to promote solubility (not to induce protein aggregation) of the protein. It should also be resistant to protease digestion. The length of the linker should be long enough to allow each domain to fold independently without any steric hindrance. It is preferred that at least one hydrophilic residue in this linker sequence be close or adjacent to the His tag to minimize the possibility of the His tag being buried within the protein core.

Biological Activity

We have found that the presence of small tags at both ends of BirA does not materially affect the biological activity of BirA as a biotin ligase. An engineered protein of the present invention that comprises two small affinity tags specifically preferred herein, namely a 53 -amino acid CBD tag and a 6-amino acid polyhistidine tag, had a higher specific activity than natural, purified BirA obtained from a commercial supplier. This shows that the BirA portion of the engineered protein of this invention retained good biological activity through the purification procedure or was in some way stabilized during the purification steps. Additionally, and advantageously, the smaller tags do not interfere with the enymatic activity of the BirA portion, and therefore the tags do not have to be cleaved from the engineered protein before assaying for enymatic activity, unlike what is sometimes necessary when larger tags are used.

Polynucleotides

h another aspect of the invention, the invention comprises polynucleotides that encode engineered proteins as described above and in particular, polynucleotides that encode the engineered protein comprising the amino acid sequence set out in Table 1 [SEQ ID NO :2]. The polynucleotides of the present invention may include, for example, unprocessed RNAs, ribozyme RNAs, mRNAs, cDNAs, genomic DNAs, B- and Z-DNAs.

A preferred polynucleotide comprises the nucleotide sequence shown in Table 1 [SEQ ID NO: 1] which encodes amino acid SEQ ID NO:2. Other polynucleotides included in the present invention encode biologically active variants of the amino acid SEQ ID NO:2.

Using the information provided herein, such as a polynucleotide sequence set out in Table 1 [SEQ ID NO:l] the portion of the polynucleotide of the invention encoding BirA may be obtained using standard screening and cloning methods. For example, to obtain a polynucleotide fragment comprising some of all of the BirA sequence, an E. coli chromosomal DNA library is probed by hybridization with a synthetic radiolabeled oligonucleotide, preferably a 17-mer or longer, that is homologous to the BirA sequence. Clones carrying DNA highly homologous to the probe are identified by using stringent hybridization conditions. By sequencing the individual clones identified by hybridization with sequencing primers designed from the sequences in the plasmid or phage DNA from which the library was constructed, it is possible to identify the BirA clones. The DNA inserts from several clones can be ligated together to obtain a full-length polynucleotide, if necessary. Suitable techniques for manipulating DNA are described by Maniatis, T., Fritsch, E. F. and Sambrook et al., MOLECULAR CLONTNG, A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). (see in particular Screening By Hybridization 1.90 and Sequencing Denatured Double- Stranded DNA Templates 13.70).

Moreover, the DNA sequence set out in Table 1 [SEQ ID NO: 1] contains an open reading frame encoding a protein having about the same number of amino acid residues set forth in Table 1 [SEQ ID NO: 2] with a deduced molecular weight that can be calculated using amino acid residue molecular weight values well known to those skilled in the art. The polynucleotide disclosed by SEQ ID NO:l, between nucleotide number 1 and the stop codon located at nucleotide number 1216 of SEQ ID NO:l, encodes the engineered protein disclosed by SEQ ID NO:2.

In a further aspect, the present invention provides for an isolated polynucleotide comprising or consisting of a polynucleotide sequence for an engineered protein which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, even more preferably at least 97-99% or 100%, to the amino acid sequence of SEQ ID NO:2, over the entire length of SEQ ID NO:2, and which exhibits biotin ligase biological activity as described above.

A polynucleotide encoding homologs and orthologs of BirA from species other than E. coli, may be obtained by a process which comprises the steps of screening an appropriate library under selected hybridization conditions, with a labeled or detectable probe consisting of or comprising the sequence of SEQ ID NO: 1 or a fragment thereof, isolating a partial or full-length clone containing said polynucleotide sequence using technques described above, and other techniques known to those of skill in the art. The invention provides a polynucleotide sequence identical over its entire length to a coding sequence (open reading frame) in Table 1 [SEQ ID NO: 1]. Also provided by the invention is a coding sequence for a mature protein or a portion thereof, by itself, as well as a coding sequence for a mature protein or a portion thereof in frame with another coding sequence, such as a sequence encoding a leader or secretory sequence, a pre-, or pro- or prepro-protein sequence. The polynucleotide of the invention may also contain at least one non-coding sequence, including for example, but not limited to at least one non-coding 5' and 3' sequence, such as the transcribed but non-translated sequences, termination signals (such as rho-dependent and rho-independent termination signals), ribosome binding sites, Kozak sequences, sequences that stabilize mRNA, introns, and polyadenylation signals. The polynucleotide sequence may also comprise additional coding sequence encoding additional amino acids.

The invention also includes a polynucleotide consisting of or comprising a polynucleotide of the formula:

wherein, at the 5' end of the molecule, X is hydrogen, a metal or a modified nucleotide residue, or together with Y defines a covalent bond, and at the 3' end of the molecule,

Y is hydrogen, a metal, or a modified nucleotide residue, or together with X defines the covalent bond, each occurrence of Ri and R is independently any nucleic acid residue or modified nucleic acid residue, m is an integer between 1 and 3000 or zero, n is an integer between 1 and 3000 or zero, and R₂ is a polynucleotide sequence or modified polynucleotide sequence of the invention, particularly SEQ ID NO:2 or variants thereof. In the polynucleotide formula above, R₂ is oriented so that its 5' end nucleic acid residue is at the left, bound to R_\, and its 3' end nucleic acid residue is at the right, bound to R₃. Any stretch of nucleic acid residues denoted by either Ri and/or R₂, where m and/or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer. Where, in a preferred embodiment, X and

Y together define a covalent bond, the polynucleotide of the above formula is a closed, circular polynucleotide, which can be a double-stranded polynucleotide wherein the formula shows a first strand to which the second strand is complementary. In another preferred embodiment m and/or n is an integer between 1 and 1000. Other preferred embodiments of the invention are provided where m is an integer between 1 and 50, 100 or 500, and n is an integer between 1 and 50, 100, or 500.

The term "polynucleotide encoding a polypeptide" as used herein encompasses polynucleotides that include a sequence encoding a protein or peptide of the invention, particularly the engineered protein, and more particularly a bacterial protein. The term also encompasses polynucleotides that include a single continuous region or discontinuous regions encoding the engineered protein (for example, polynucleotides interrupted by integrated phage, an integrated insertion sequence, an integrated vector sequence, an integrated transposon sequence, or due to RNA editing or genomic DNA reorganization) together with additional regions, that also may contain coding and/or non-coding sequences.

Preferred embodiments are polynucleotides encoding proteins that retain substantially the same biological function or activity as the engineered protein encoded by a DNA of Table 1 [SEQ ID NO :2]. Further particularly preferred embodiments are polynucleotides encoding variants of the engineered protein, in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, modified, deleted and/or added, in any combination. Especially preferred among these are silent substitutions, additions and deletions, that do not alter the properties and activities of the engineered protein.

Other preferred embodiments of this invention include polynucleotides that hybridize, particularly under stringent conditions, to polynucleotides described herein including polynucleotide sequences encoding the engineered protein. As herein used, the terms "stringent conditions" and "stringent hybridization conditions" mean hybridization occurring only if there is at least 95% and preferably at least 97% identity between the sequences. A specific example of stringent hybridization conditions is overnight incubation at 42° C in a solution comprising: 50% formamide, 5xSSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 micrograms/ml of denatured, sheared salmon sperm DNA, followed by washing the hybridization support in O.lxSSC at about 65° C. Hybridization and wash conditions are well known and exemplified in Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), particularly Chapter 11 therein. Solution hybridization may also be used with the polynucleotide sequences provided by the invention.

There are several methods available and well known to those skilled in the art to obtain full-length DNAs, or extend short DNAs, for example those based on the method of Rapid Amplification of cDNA ends (RACE) (see, for example, Frohman, et al., PNAS USA 85: 8998-9002, 1988). Recent modifications of the technique, exemplified by the Marathon™ technology (Clontech Laboratories Inc.) for example, have significantly simplified the search for longer cDNAs. In the Marathon™ technology, cDNAs have been prepared from mRNA extracted from a chosen tissue and an ' adaptor" sequence ligated onto each end. Nucleic acid amplification (PCR) is then carried out to amplify the "missing" 5' end of the DNA using a combination of gene specific and adaptor specific oligonucleotide primers. The PCR reaction is then repeated using "nested" primers, that is, primers designed to anneal within the amplified product (typically an adaptor specific primer that anneals further 3' in the adaptor sequence and a gene specific primer that anneals further 5' in the selected gene sequence). The products of this reaction can then be analyzed by DNA sequencing and a fill- length DNA constructed either by joining the product directly to the existing DNA to give a complete sequence, or carrying out a separate full-length PCR using the new sequence information for the design of the 5' primer.

The invention also provides polynucleotides that encode a polypeptide that is the engineered protein plus additional amino or carboxyl-terminal amino acids, or amino acids interior to the engineered protein (when the mature form has more than one polypeptide chain, for instance). Such sequences may play a role in processing of a protein from precursor to a mature form, may allow protein transport, may lengthen or shorten protein half-life or may facilitate manipulation of a protein for assay or production, among other things. As generally is the case in vivo, the additional amino acids may be processed from the engineered protein by cellular enzymes.

For each and every polynucleotide of the invention there is provided a polynucleotide complementary to it. It is preferred that these complementary polynucleotides are fully complementary to each polynucleotide with which they are complementary.

In addition to the standard A, G, C, T/U representations for nucleotides, the letter "N" may also be used in describing a nucleotide. "N" means that any of the four DNA or RNA nucleotides may appear at such a designated position in the DNA or RNA sequence, except it is preferred that N is not a nucleic acid that, when taken in combination with adjacent nucleotide positions, when read in the correct reading frame, would have the effect of generating a premature termination codon in such reading frame.

In sum, a polynucleotide of the invention may encode the engineered protein, the engineered protein plus a leader sequence (which may be referred to as a preprotein), or a precursor of the engineered protein, having one or more prosequences that are not the leader sequences of a preprotein, or a preproprotein, which is a precursor to a proprotein, having a leader sequence and one or more prosequences, which generally are removed during processing steps that produce active and mature forms of the polypeptide.

Vectors, Host Cells, Expression Systems

The invention also relates to vectors that comprise a polynucleotide or polynucleotides of the invention, host cells that are genetically engineered with vectors of the invention and the production of engineered proteins of the invention by recombinant techniques. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the invention. Engineered proteins of the present invention may be prepared by processes known to those skilled in the art, from genetically engineered host cells comprising expression systems. Accordingly, in a further aspect, the present invention includes expression systems which comprise a polynucleotide or polynucleotides of the present invention, to host cells which are genetically engineered with such expression systems, and to the production of engineered proteins of the invention by recombinant techniques.

For production of the engineered proteins of the invention, host cells can be genetically engineered to incorporate expression systems or portions thereof or polynucleotides of the invention. Introduction of a polynucleotide into the host cell can be effected by methods described in standard laboratory manuals, such as Davis, et al., BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), such as, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction and infection.

Representative examples of appropriate hosts include bacterial cells, such as cells of streptococci, staphylococci, enterococci E. coli, streptomyces, cyanobacteria, Bacillus subtilis, and Streptococcus pneumoniae; fungal cells, such as cells of a yeast, Kluveromyces, Saccharomyces, a basidiomycete, Candida albicans and Aspergillus; insect cells such as cells of Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293, CV-1 and Bowes melanoma cells; and plant cells, such as cells of a gynmosperm or angiosperm.

A great variety of expression systems can be used to produce the engineered proteins of the invention. Such vectors include, among others, chromosomal-, episomal- and virus-derived vectors, for example, vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses, picornaviruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. The expression system constructs may contain control regions that regulate as well as engender expression. Generally, any system or vector suitable to maintain, propagate or express polynucleotides and/or to express a protein in a host may be used for expression in this regard. The appropriate DNA sequence may be inserted into the expression system by any of a variety of well- known and routine techniques, such as, for example, those set forth in Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, (supra).

In recombinant expression systems in eukaryotes, for secretion of a translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretion signals may be incorporated into the expressed protein. These signals may be endogenous to the protein or they may be heterologous signals.

In a preferred embodiment, the engineered protein, and in particular CBD- BirA-His, may be expressed intracellularly using IPTG induction and a pET-29b based vector in E. coli. However, as described below, a large proportion of CBD- BirA-His accumulates as inclusion bodies at a growth temperature of 30° C. The applicants have found that by supplementing the culture media with biotin and by lowering the growth temperature to about 25° C (post-IPTG induction), solubility of CBD-BirA-His could be greatly increased. Therefore, supplementation of biotin in the culture medium could help reduce the formation of inclusion bodies. Biotin was commonly included in the culture medium in in vivo biotinylation studies involving the E. coli system since E. coli has been shown to uptake biotin via an active transport mechanism (31). In those studies, biotin served mainly as one of the substrates for BirA in the biotinylation reaction. We have found that biotin can possibly enhance the proper folding of BirA in favour of soluble protein formation. Engineered proteins of the invention can be recovered and purified from recombinant cell cultures by metal chelation chromatography and/or chitin affinity chromatography, which are well known techniques in the art. In a preferred embodiment, a Ni²⁺ chelation column may effectively bind CBD-BirA-His and reasonably pure fractions (>80%) may be recovered by elution with imidazole. Higher purities may be achieved by repeated reloading the purified material to the chelation column. A chitin affinity scheme may be more efficient. A single column operation may produce CBD-BirA-His with over 95% purity, however, a significant portion of the protein tends to be retained on the column and cannot be recovered even with extensive washes and elutions. Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification.

Methods of using the Polypeptides

To capture the full advantages of in vitro biotinylation, a ready source of easily purified, high quality BirA is needed. The present invention attempts to address this concern by providing an engineered E. coli BirA with a different tag at each end (CBD-BirA-His). These tags enable easy recovery of the protein by simple column manipulations. Use of the His-tag allows a one-step recovery of large amounts of reasonably pure CBD-BirA-His, while use of the CBD tag enables, again, a single-column recovery of a lesser quantity of ultrapure CBD-BirA-His. These two grades of CBD-BirA-His can be found useful in different applications. For example, reasonably pure CBD-BirA-His can be used to biotinylate a crude extract (such as the secreted fraction) as other contaminants can be removed later via the monomeric avidin step. On the other hand, ultrapure CBD-BirA-His is critical in the biotinylation of pure proteins (such as affinity-purified single chain antibodies). Besides the tag advantage, the production yield and quality of our engineered BirA compare favourably with the literature data. By supplementing the medium with biotin and lowering the post-induction temperature to 25°C, the soluble CBD-BirA-His reached a level of 100 mg per liter of culture. This level is double the amount of GST-BirA reported previously (30). Moreover, the specific activity of CBD-BirA-His was found to be more than that of the natural BirA from a commercial source. In one study involving GST-BirA (30), thrombin was applied to cleave off GST from the fusion and the resulting BirA showed a comparable activity similar to that of the wild type BirA. In another case (19), GST-BirA, used uncleaved, was shown to retain biotin ligase activity but the specific activities of the fused and non-fused versions were not studied.

Purification of Proteins

The engineered proteins of the present invention, and CBD-BirA-His in particular, are useful in methods involving in vitro biotinylation to recover or purify intracellular and secretory proteins. Generally, the engineered proteins of the present invention may be used to biotinylate a protein, either a native protein or one to which a biotinylation tag has been added in vitro. The biotinylated protein may then be recovered or purified using a monomeric avidin or nitro-avidin column.

In vitro biotinylation offers a general tool for affinity purifying secretory proteins not only from E. coli but also from other organisms such as B. subtilis. This approach is most valuable for the purification of proteins which cannot be recovered by other affinity purification methods and which require multiple chromatographic steps for their purification. As described herein, addition of the biotinylation tag to staphylokinase affected neither the production yield nor the biological activity of staphylokinase and intact SAK-PFB could be produced as confirmed by mass spectrometric analysis. This system works best when the target protein has a high-level expression, the fusion is stable, and protease activity is absent. The high efficiency biotinylation achieved with our SAK-PFB study may be attributed to the remarkable secretory yield of SAK in B. subtilis (over 100 mg/1 in a shake flask) (33), the stability of S AKPFB, and the use of an eight-protease deficient strain which has been shown to dramatically enhance the yield (24) and stability (unpublished data) of some secretory proteins in B. subtilis. The high efficiency biotinylation, coupled with the high capacity of monomeric avidin with its exceptional affinity and specificity to biotin, contributes to a remarkable recovery of quantitative amounts of distinctly pure staphylokinase. This approach can be applied to other secretory proteins from B. subtilis.

Therefore, in one aspect, the invention comprises a method of purifying Bacillus subtilis staphylokinase. Staphylokinase (SAK) is a promising blood clot dissolving agent (29). A biotinylation tag such as PFB may be added to the C- terminal end of SAK without affecting secretory production yield. SAK-PFB may then be biotinylated in a concentrated culture supernatant using the engineered proteins of the present invention. The biotinylated SAK-PFB may then be effectively purified using a monomeric avidin agarose column. We have found that biotinylated SAK-PFB purified by this method retains full biological activity.

One skilled in the art may apply this in vitro biotinylation using the engineered proteins of the present invention to a wide range of biomolecules to purify the biomolecule. It is especially preferred for secretory proteins which are difficult to purify using conventional techniques.

Besides protein purification, the homogeneous biotinylated products made possible by the highly selective, site-specific action of CBD-BirA-His on the biotinylation tag offers many other applications. They may serve as agents in immunoassays, drug delivery, imaging and targeting (34, 35, 36, 37). Biotinylated proteins can also be immobilized in an orientation-specific manner (38) to generate protein or antibody biochips for surface plasmon resonance based biosensor measurements (39, 40), active electronic microchips for biomolecule detection and quantification (41), and high density protein microarrays for high throughput proteomics studies (42).

Two interesting observations were also made during the purification of the biotinylated proteins. Occasionally, we detected a biotin-BirA complex in Western blot probed with streptavidin-horseradish peroxidase even though the sample had been boiled in the presence of SDS before loading to the SDS-polyacrylamide gel. This complex is likely to be the tight entity (Ka = 7 x 10^"11) formed between BirA and biotinoyl-5'-AMP, an intermediate in the biotinylation reaction carried out by BirA (32). The presence of this complex means that postbiotinylation removal of BirA is necessary not only when pure target protein is involved but also when crude sample is used for biotinylation. The installation of the N-terminal CBD in CBD-BirA-His allows rapid removal of BirA by the use of chitin beads. In the purification of SAK- PFB, CBD-BirA-His was removed by chitin bead treatment in a simple centrifugation step to avoid the potential problem of contamination. Thus, the tags on CBD-BirA-His facilitate not only purification of CBD-BirA-His but also removal of CBD-BirA-His from the postbiotinylation reaction mixture. Another interesting observation is that the biotinylated protein exhibited a small mobility shift on the SDS gel. This has a practical application for the biotinylation of small target proteins as one may be able to monitor the extent of biotinylation, easily by SDS-PAGE. This method worked well for SAK-PFB with a molecular mass of 19 kDa.

EXAMPLES

The examples below are carried out using standard techniques, which are well known and routine to those skilled in the art, except where otherwise described in detail. These examples are intended to be illustrative, but not limiting, of the invention.

Construction of pET-BirA-His

Plasmid pET-BirA-His is an expression vector that produces BirA with a C-terminal hexahistidine tag in E. coli using the T7 promoter system. E. coli BirA was amplified by PCR with E. coli genomic DNA as the template and synthetic oligonucleotides ECBIRAF (5' GGGAATTCTAAGGATAACACCGTGCCACTG 3' [SEQ ID NO:3]) and ECBIRAB (5' GGAAGCTT

TTGTTTTTCTGCACTACGCAGGG 3'[SEQ ID NO:4]) as the forward and reverse primers, respectively. The amplified product carried an EcόRl site at the 5' end and a Hindm site at the 3' end. The 970-bp fragment was digested with Ecό IHindSl and inserted in frame to pET-29b (Novagen, USA) to give pET-BirA-His. Construction of an Expression Vector Comprising a BirA Insert

E. coli BirA can be amplified by PCR with E. coli genomic DNA as the template and synthetic oligonucleotides comprising SEQ ID NO: 3 and SEQ ID NO: 4 as the forward and reverse primers, respectively. The amplified product will carry an -ZicoRI site at the 5' end and a HindSl site at the 3' end. The 970-bp fragment can then be digested with EcoKHHinaT l and inserted in frame into any one of a number of expression vectors, known to those skilled in the art.

Construction of pET-CBD-BirA-His

This vector allows the production of CBD-BirA-His in E. coli. The gene encoding a chitin binding domain (22) was amplified from the pC YBI plasmid carrying the CBD of chitinase Al (New England BioLabs, Canada) using the forward primer CBDF (5' CCCATATGACGACAAATCCTGGTGTATCC 3'[SEQ ID NO:5]) and the backward primer CBDB (5'

CCAGATCTTGAAGCTGCCACAAGGCAGGAAC 3'[SEQ ID NO:6]). The 165-bp amplified product was then digested by Ndel and BgM and inserted into pET-BirA-His. The resulting plasmid, designated pET-CBD-BirA-His, was transformed to E. coli BL21(DE3) (Novagen, USA) for expression studies.

The restriction map of pET-CBD-BirA-His is shown in Figure 7 and the complete pET-CBD-BirA-His DNA sequence [SEQ ID NO:7] is shown in Figure 8.

Construction of an Expression Vector Comprising a CBD-BirA Insert

The expression vector comprising a BirA insert can be modified to include CBD, using procedures analogous to those described above for constructing pET-CBD-BirA-His. Specifically forward and reverse PCR primers identical to SEQ ID NO:5 and SEQ ID NO:6 may be used if the expression vector that comprises the BirA insert will allow in frame insertion into the vector, using Ndel and BgM. Alternatively forward and reverse PCR primers similar to SEQ ID NO:5 and SEQ ID NO: 6, but differing in the restriction endonuclease recognition sequences, so as to be compatible with restriction sites in the expression vector that comprises the BirA insert, to allow an in-frame insertion, can be designed and used, using procedures known to those skilled in the art.

Construction of pSAKPFB

This is a B. subtilis vector for secretory production of staphylokinase (SAK) containing a Cterminal biotinylation peptide (PFB). This vector used a strong and constitutively expressed promoter (P43) to drive the transcription and a B. subtilis levansucrase signal peptide to direct the secretion. The biotinylation tag was fused translationally to secretory staphylokinase in the following manner. The sequence encoding PFB was fused in frame to the 3'-end of the sak gene in pSAK-Kl , a pWB980-based vector in B. subtilis (23), by PCR with pSAK-Kl as the template, 5' CAAGCAACAGTATTAACC 3' [SEQ ID NO: 8] as the forward primer and 5' CCAAGCTTATCGATGATTCCAAACCATTTTTTGTGCAT CAAGAATATGATGAAGGGATCCAGAGCCACTAGTAGATCC 3' [SEQ ID NO:9]as the backward primer. The backward primer encodes a 15-amino acid peptide with the amino acid sequence LHHILDAQKMVWNHR [SEQ ID NO: 10] (11). The amplified 578-bp fragment was digested with HindRI and used to replace an equivalent fragment from HinaTI digested pSAK-Kl. The resulting plasmid, designated pSAKPFB, was transformed to B. subtilis WB800, an eight-protease deficient strain (24) and the transformants screened for the right orientation of the insert.

Cell growth

E. coli BL21 [pET-CBD-BirA-His] was grown at 30°C in Luria broth (1% tryptone, 0.5% yeast extract, 0.2% NaCl) containing 30 μg/ml kanamycin to 150 klett units in a shake flask. IPTG was then added to a final concentration of 0.1 mM and growth continued for 5-10 hours. Cell density was measured using a Klett-Summerson photoelectric colorimeter with a green filter (Klett Mfg. Co., USA). B. subtilis

WB800[pSAKPFB] was cultivated in super-rich medium (25) containing 10 pg ml of kanamycin at 37°C in a shake flask. Cells were harvested at 5-6 hours after inoculation.

Purification of BirA

Cells of E. coli BL21 [pET-CBD-BirA-His] were harvested by centrifugation at 10,000 x g for 5 min at 4°C. Cell pellet was resuspended in lysing buffer, disrupted with French press and the crude lysate was separated into the soluble and insoluble fractions by centrifugation (20,000 x g for 20 min). CBD-BirA-His in the soluble fraction was purified by either of two schemes: metal chelation chromatography or chitin affinity chromatography. For metal chelation chromatography, the lysing buffer contained 15 mM imidazole, 0.5M NaCl, 0.1% Triton X, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 20 mM Tris-HCl, pH 8.0. His.Bind Quick 900 cartridges (Novagen, USA) charged with Ni²⁺ were used as the affinity matrix. CBD-BirA-His was eluted stepwise with increasing imidazole concentrations (60 mM, 250 mM, 1 M imidazole) according to the manufacturer's suggestions. For chitin afflinity chromatography, cells were lysed in buffer containing IM NaCl, 1 mM EDTA, 0.1% Triton X, 5 mM β-mercaptoethanol, 1 mM PMSF and 20 mM sodium phosphate, pH 7.0. The soluble cellular fraction was loaded to a column packed with chitin beads (New England BioLabs, Canada) equilibrated in lysing buffer. After washing with 5-10 column volumes of lysing buffer followed by 20 mM sodium acetate, pH 5.5, CBD-BirA-His was eluted with 20 mM acetic acid, pH 3.0.

In either scheme, fractions containing pure CBD-BirA-His (confirmed by

SDS-PAGE) were pooled, concentrated and buffer-changed to a storage solution containing 50 mM imidazole, 50 mM NaCl, 5% glycerol and 5 mM β-mercaptoethanol, pH 6.8, using Ultrafree-4 centrifugation tubes (Millipore Corporation, USA). Pure CBD-BirA-His was quantified by its absorbance at 280 nm using a molar extinction coefficient of 68420 M ^_1 cm ^-1 (26). Purification of SAK-PFB

Culture supernatant of B. subtilis WB800[pSAKPFB] was separated from the cells by centrifugation at 10,000 x g for 10 min at 4°C. SAK-PFB was precipitated with ammonium sulfate to 65% saturation at 4°C, desalted by dialysis, concentrated to appropriate volume and buffer-changed to 10 mM Tris-HC 1 , pH 8.0, using

Ultrafree-4 centrifugation tubes (Millipore Corporation, USA). The sample was then biotinylated at 30°C for 4 hours to overnight, using CBD-BirA-His. The reaction mixture contained 50 mM bicine (pH 8.3), 10 mM ATP, 10 mM magnesium acetate, 50 μM biotin, and for every ml of final mix, 500 μg of SAK-PFB and 5 μg of purified E. coli CBD-BirA-His. Following the reaction, the sample was mixed with a small amount of chitin beads to remove CBD-BirA-His. After a simple centrifugation to remove the chitin beads, the sample was passed over a column containing Sephadex G-25 (Amersham Pharmacia Biotech, Canada) to remove the excess biotin. Biotinylated SAK-PFB was separated the unbiotinylated proteins by passing the sample over a monomeric avidin agarose column (Pierce, USA). Bound biotinylated SAK-PFB was eluted by competition with 2 mM d-biotin. Pure biotinylated SAK-PFB, was quantified by its absorbance at 280 nm using a molar extinction coefficient of 22,900 M^cm^"1 (26) for calculation.

Determination of the activity of purified CBD-BirA-His

The activity of purified CBD-BirA-His was compared with that of a wild type E. coli BirA available from a commercial source (Avidity, USA) using an ELISA method (13). In this assay, maltose binding protein- AviTag fusion (MBP-AviTag, Avidity, USA) was used as the substrate. AviTag is a peptide tag for efficient biotinylation (11). MBP-AviTag was adsorbed to the wells of a Reacti-bind maleic anhydride activated polystyrene strip plate (Pierce, USA). Biotinylation was carried out at 30°C with different amounts of enzymes and different reaction times. The reaction mixture contained 50 mM bicine (pH 8.3), 10 M ATP, 10 mM magnesium acetate, 50 μM biotin and BirA from different sources. Biotin ligated to the AviTag was detected by its interaction with streptavidin-horseradish peroxidase (Pierce, USA) using 1 step slow TMBELISA (3,3',5,5'-tetramethylbenzidine, Pierce) as the color development reagent. A standard curve of biotinylation reaction was established using known quantities of fully biotinylated MBP-AviTag (Avidity, USA). Readings were taken at end point at 450 nm using a Bio-Tek CERES 900 plate reader (Bio-Tek Insiruments, Inc., USA).

Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometric analyses

Protein samples in 25 mM ammonium acetate and the matrix solution of sinapinic acid were mixed on the MALDI plate and analyzed on a Perseptive

Biosystems (Framingham Mass.) Voyager-DE STR Mass spectrometer equipped with a pulsed nitrogen laser operated at 337 nm in a linear mode. The mass spectrometer was previously calibrated with apomyoglobin (horse skeletal) m/z 16952.56 and its dimer m/z 33905.12. These analyses were done in Plant Biotechnology Institute, National Research Council of Canada, Saskatoon, Canada.

Other methods

Vent DNA polymerase (New England BioLabs, Canada) was used for all DNA amplification reactions. The sequence of all PCR products was confirmed to be free of PCR errors by nucleotide sequencing based on the dideoxy method using a T7 sequencing kit from Amersham Pharmacia Biotech, Canada. SDS-polyacrylamide gel electrophoresis followed standard procedure based on the Laenimli system. Western blot was done on a nitrocellulose membrane using 4-chloro- 1 -naphthol (Bio-Rad, Canada) as the color development reagent. SAK activity was determined by radial caseinolysis assay on plasminogen-skim milk agarose plate (27).

Production of E. coli CBD-BirA-His using the pET expression system. An IPTG-induced expression of BirA in a pET-29b based vector was used for intracellular production ofE. coli CBD-BirA-His in BL21(DE3). CBD-BirA-His (with both

CBD- and His-tags) produced migrated as a 40-kDa protein on the SDS gel (Fig. 1). The presence of the His-tag was found to complicate the production because, at a growth temperature of 30°C, 90% of CBD-BirA-His accumulated as inclusion bodies (Fig. 1A, lanes 1 and 2). In contrast, over 80% of CBD-BirA (no His tag) produced under the same cultivation condition was in the soluble form (data not shown). To address the solubility problem, different measures were taken. These include lowering the growth temperature from 30°C downwards, lowering the salt concentration in the culture medium, varying the IPTG levels, and modifying the cellular osmotic environment with the use of sorbitol and betaine during cell growth (28). These measures at best yielded marginal improvement with still more than 70% of CBD-BirA-His present as insoluble aggregates. However, supplementing the culture medium with 10-20 μM biotin not only enhanced the growth rate of the culture (data not shown) but also conspicuously promoted solubility of CBD-BirA-His with about 40% of the protein in the soluble fraction (Fig. 1 A, lanes 3 and 4; Fig. 1 B, lanes 1 and 2). Moreover, whereas temperature lowering by itself did not effectively solve the problem of inclusion body formation, a measure combining biotin supplementation and temperature lowering (25°C, post-induction) enhanced BirA solubility significantly. Typically, 70-90% of BirA produced under this condition was in the soluble form (Fig IB, lanes 3 and 4), amounting to about 100 mg of soluble CBD-BirA-His per liter of culture.

Purification of Engineered E. coli BirA

CBD-BirA-His was equipped with two tags: a 6-amino-acid histidine tag preceded by an 8amino-acid linker and a 53-amino-acid chitin binding domain followed by an 18-amino-acid linker. These tags allow rapid purification of the protein by either scheme: metal chelation or chitin affinity chromatography. Fig. 2A shows the purification of CBD-BirA-His using a Ni²⁺ chelation column.

CBD-BirA-His bound to the column effectively with essentially no loss in the flow-through fractions (lane 2). Reasonably pure fractions (over 80% purity) were recovered by elution with imidazole (lanes 3-5). CBD-BirA-His in these fractions could be further purified to over 95% purity by repeatedly reloading the purified CBD-BirA-His to the Ni²⁺ chelation column. Tlie chitin affinity scheme was more efficient. CBD-BirA-His bound to the chitin column with high affinity and great specificity with no CBD-BirA-His detectable in the flowthrough and washes (Fig. 2B, lanes 2 and 3). A single-column operation was usually adequate to recover CBD-BirA-His with over 95% purity (Fig. 2B, lane 4). Chitin affinity chromatography, however, has a major drawback. About 40-50% of the CBD-BirA-His tended to be retained on the column and could not be recovered even with extensive washes and elutions at low pH. Despite this drawback, we have been able to recover 1.5-2 mg of highly pure CBD-BirA-His from 100 ml of shake flask culture using the chitin column, representing an overall recovery yield of 15-20%. The recovery rate with the metal chelation scheme (involving three cycles of Ni' chelation column) to purify CBD-BirA-His with over 95% purity is similar.

Purified engineered BirA demonstrated high biological activity

Activity of CBD-BirA-His was determined by its ability to biotinylate maltose binding protein tagged with a short biotinylation peptide designated AviTag (11) in an ELISA study. With unbiotinylated MBP-AviTag as the substrate using parameters (amount of enzyme used and reaction time) that ensured a linear rate of enzymatic reaction, the activity of CBD-BirA-His purified from either scheme was found to be 50% more active than that of the natural E. coli BirA from a commercial source (Table 2).

Table 2. Activity of BirA from different sources

Activity of BirA was determined by ELISA method (13) using unbiotinylated

MBP-AviTag (Avidity, USA) as the substrate. Specific activity of BirA is defined as ng biotinylated MBP AviTag formed per min per μg of enzyme at 30°C. ¹ CBD-BirA-His purified by metal chelation chromatography. ²CBD-BirA-His purified by chitin affinity scheme. ³Wild type E. coli BirA obtained from a commercial supplier (Avidity, USA). Data represent the average of two independent trials.

This shows that the CBD-BirA-His produced and purified in accordance with the present invention may be of high quality. The presence of His-tag has little effect on the biological activity of the purified enzyme as CBD-BirA and CBD-BirA-His exhibited similar specific activities on biotinylation of MBP-AviTag (data not shown). The readiness of CBD-BirA-His to biotinylate proteins with a biotinylation tag was also demonstrated in a Western blot analysis (Fig. 3). Two test proteins were used as examples: MBP-AviTag and staphylokinase tagged with another biotinylation tag designated PFB. Probing with streptavidin-horseradish peroxidase showed biotinylation of both proteins with BirA (Fig. 3B, lanes 1 and 2).

Engineered BirA is active in a fairly broad pH range

The pH activity profile of CBD-BirA-His was established with an ELISA study similar to the one used for the determination of its biotinylation activity.

Different reagents were used to provide buffering capacity for a broad pH range (see legend to Fig. 4). MBP-AviTag was used as the substrate. Fig. 4 shows that CBD-BirA-His had a pH optimum around 6.5. It retained a fairly high activity at pH 5.5-8.3, but the activity dropped substantially at either ends. This information would be useful for one to tailor an optimal condition for in vitro biotinylation with this enzyme. To our knowledge, the pH activity profile of natural E. coli BirA has not been systematically studied before.

Secretory production of staphylokinase-PFB from B. subtilis To explore the possibility of purifying a secretory fusion protein carrying a biotinylation tag from a B. subtilis culture supernatant via in vitro biotinylation using the engineered BirA, staphylokinase (SAK), a very promising blood clot dissolving agent (29), was used as a model system. A 15-amino-acid biotinylation tag (PFB) was added to the C-terminal end of SAK containing an 18-amino-acid C-terminal linker sequence [(GSTSG)₃SGS] . Addition of the linker and the biotinylation tag did not affect the secretory production yield of SAK-PFB since SAK with or without PFB was produced at a comparable level (Fig. 5 A, lanes 1 and 2). When analyzed by SDS-PAGE, SAK-PFB showed an apparent molecular mass of 21 kDa. The calculated molecular mass of SAK-PFB is 18,862 Da. To confirm that the intact form of SAK- PFB was produced from B. subtilis, the molecular mass of SAK-PFB was determined by MALDI-TOF mass spectrometry. The observed molecular mass matched closely with the expected value and was determined to be 18,861.22 Da (data not shown).

Functional SAK-PFB could be purified via in vitro biotinylation using the engineered BirA

After concentrated from the culture supernatant, SAK-PFB was biotinylated in vitro using purified CBD-BirA-His. The rate of biotinylation depends, among other variables, on the amount of enzyme used for the reaction. As SAK-PFB is fairly stable, biotinylation could be carried out using varying amounts of enzyme from several hours to overnight with no apparent adverse effect. Biotinylated SAK-PFB, with an apparent molecular mass of 21.5 kDa on tlie SDS gel, migrated more slowly than its unbiotinylated counterpart (Fig. 5A, lane 3 vs. lane 2, Fig. 6, lane 2 vs. lane 1). This allows us to easily monitor the extent of biotinylation. In all biotinylation runs attempted so far, over 95% biotinylation of SAK-PFB could be achieved as demonstrated by the absence of any significant amount of SAK-PFB in the flow-through or washes of the monomeric avidin agarose column (Fig. 6, lanes 3 and 4). The completion of biotinylation was also demonstrated by the MALDI-TOF mass spectrometric analysis. The peak with the expected molecular mass corresponding to the unbiotinylated form of SAK-PFB disappeared completely in the biotinylated sample while a new peak with the expected molecular mass corresponding to the biotinylated form appeared (data not shown). Biotinylated SAK-PFB could be effectively purified using a monomeric avidin agarose column with remarkable specificity (Fig. 6, lanes 5-7). We have been able to recover about 450 μg of highly pure SAKPFB from a crude sample containing 600 μg of SAK-PFB on a single column, representing an overall yield of 75%. SAKPFB purified by this method showed full biological activity as compared with both the unbiotinylated form and the natural, untagged SAK on a plasminogen assay ml of B. subtilis culture.

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Claims

WHAT IS CLAIMED IS:

I . An engineered protein comprising a biologically active biotin ligase and a chitin binding domain.

2. The protein of claim 1 further comprising a polyhistidine tag.

3. The protein of claim 2 wherein the chitin binding domain is attached to the N-terminal of the biotin ligase and the polyhistidine tag is attached to the C-terminal of the biotin ligase.

4. The protein of claim 2 wherein the polyhistidine tag is hexahistidine.

5. The protein of claim 1 or 2 wherein the biotin ligase is BirA.

6. The protein of claim 5 having the amino acid sequence SEQ ID NO: 2.

7. A polynucleotide which encodes for a polypeptide of claim 1, 2, 3, 5 or 6.

8. The polynucleotide of claim 7 comprising the nucleotide sequence SEQ ID NO:2.

9. An expression vector comprising the polynucleotide of claim 7 or 8.

10. The expression vector of claim 9 comprising pET-CBD-BirA-His.

II. A host cell comprising the expression vector of claim 9 or 10.

12. The host cell of claim 12 which is E. coli BL21.

13. A method of producing a polypeptide of claim 1 comprising the steps of introducing an expression vector of claim 9 into a E. coli BL21 host cell and culturing the host cell at a temperature less than 30° C and in the presence of biotin.

14. The method of claim 10 wherein the host cell is cultured at a temperature of about 25° C and in a culture medium containing biotin in a concentration from about 10 uM to about 20 uM.

15. A method of producing and recovering a protein comprising the steps of:

(a) culturing cells which express the protein tagged with a biotinylation peptide;

(b) biotinylating the protein with an engineered protein of claim 1 ;

(c) separating the biotinylated protein by affinity chromatography.

16. The method of claim 15 wherein the polypeptide is removed post- biotinylation using chitin beads.

17. The method of claim 15 or 16 wherein the protein is a secretory protein.

18. The method of claim 17 wherein the protein is staphylokinase.