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WO1999037785A1 - PROTEINE BirA RECOMBINANTE - Google Patents

PROTEINE BirA RECOMBINANTE Download PDF

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Publication number
WO1999037785A1
WO1999037785A1 PCT/GB1999/000245 GB9900245W WO9937785A1 WO 1999037785 A1 WO1999037785 A1 WO 1999037785A1 GB 9900245 W GB9900245 W GB 9900245W WO 9937785 A1 WO9937785 A1 WO 9937785A1
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Prior art keywords
bira
fusion protein
protein
recombinant
enzyme
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Ceased
Application number
PCT/GB1999/000245
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English (en)
Inventor
Christopher Anthony O'callaghan
Bent Karsten Jakobsen
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Oxford University Innovation Ltd
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Oxford University Innovation Ltd
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Publication of WO1999037785A1 publication Critical patent/WO1999037785A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)

Definitions

  • This invention relates to a method for the production of recombinant BirA protein and to a vector encoding the protein.
  • the E. coli repressor of biotin biosynthesis (BirA) is an enzyme which plays an important role in the biotin regulatory system of this bacterium.
  • a function of the enzyme is the transfer of biotin to a unique lysine residue on the acceptor protein, the biotin carboxyl carrier protein subunit of acetyl-CoA carboxylase, which possesses a target sequence of amino acids.
  • the biotin carboxyl carrier protein sequence was first described by Sutton et_aj in J. Biol. Chem. (1977) 252: 3934-3940.
  • the amino acid sequence of the protein as given in GenBank M80458 is
  • the BirA enzyme can be used to specifically transfer a biotin group to a protein possessing a target amino acid sequence such as LHHILDAQKMVWNHR [SEQ ID NO:1]. Further biotinylation target sequences are described for example in WO 95/04069, US 5,723,584, US 5,487,993 and EP 0550693. Thus by incorporating one of these sequences, or functionally equivalent sequences known in the art, into a protein using genetic engineering, BirA can be used to specifically label the engineered protein with biotin.
  • Biotin labelling is useful for many purposes, for instance a biotinylated protein can be separated from other unlabelled proteins by binding it to streptavidin.
  • biotinylate proteins the methods used are non- specific and usually result in biotin molecules being randomly attached at numerous sites all over the protein.
  • the orientation of the protein when attached to streptavidin is unknown and in practice this can be a major problem.
  • Artificial tetramers or other multimeric complexes of a biotinylated protein can be made by binding biotinylated monomers to streptavidin (1) which possesses four binding sites for biotin.
  • Such tetramers are useful for staining and detection as their increased potential binding avidity will increase the strength of any interaction with molecules of interest (US 5,635,363).
  • the inventors have produced class 1 a and 1 b MHC molecules which have a BirA recognition sequence allowing their specific biotinylation. This, in turn, facilitates attachment of the MHC molecule to streptavidin which itself has been attached to a fluorescent label.
  • These tetramers can be used to identify cells of the immune system which are specific for the MHC molecules and therefore may be of great use as diagnostic and research tools.
  • biotinylated proteins Numerous other applications for biotinylated proteins are described in the literature, for example O'Callaghan et al (1999) Anal. Biochem. 266: 9-15. A variety of detection reagents, systems and kits are available for the detection of biotinylated proteins. Specific biotinylating enzyme such as BirA will be useful in all of these.
  • the BirA protein which is also a transcriptional repressor protein, is normally expressed at low levels in E. coli.
  • Present methods of increasing expression and associated purification schemes are inefficient.
  • One method (2) gives a moderate amount of expression of the BirA protein (0.5-1 .0% of the total cellular protein) and employs a three chromatography matrix purification procedure.
  • Another method (3) produces almost 100% active protein, at a similarly low level and also requiring three chromatographic matrices in the purification procedure.
  • BirA is not easily produced in large quantities and is expensive to purchase. Better methods of expressing and purifying BirA, and of producing enzyme of high quality are required.
  • the inventors have devised a method of producing and purifying BirA which gives unexpected increases over the yield of other methods and simultaneously decreases the time and cost of purification considerably.
  • the coding sequence of BirA is fused to a coding sequence for a tag such as glutathione-S-transferase expression and recovery levels are greatly improved.
  • This phenomenon forms the basis of the invention.
  • the BirA protein has a transcriptional repressor function in E. coli and as such is not likely to be a suitable candidate for the high-level artificial expression obtained by the inventors.
  • Several attempts were made by the inventors to over-produce the protein using methods similar to those published (2,3), all of which resulted in poor expression.
  • the invention provides in one aspect a method for the production of a recombinant BirA enzyme which comprises the steps of: i) providing an expression vector containing an expression regulation sequence operably linked to a sequence encoding a fusion protein of a tag domain and a BirA enzyme; ii) transforming cells of a suitable bacterial host strain with the expression vector, culturing said cells under conditions to allow expression of the fusion protein, and obtaining from said cells an extract comprising the fusion protein; iii) performing at least one separation step by bringing said extract into contact with a binding reagent on a solid phase, said binding reagent capable of binding to the tag domain of the fusion protein, and removing unbound components of the extract; iv) removing recombinant BirA enzyme from the solid phase.
  • the recombinant BirA enzyme is removed from the solid phase by cleaving the recombinant BirA enzyme of the fusion protein from the tag domain.
  • cleavage is brought about by enzymatic means, such as by the action of thrombin on a thrombin cleavable site.
  • Other protease/protease recognition sequence combinations known in the art, such as the Factor Xa, or the PreScissionTM Protease (Amersham-Pharmacia Biotec) systems are also envisaged for providing a means of cleaving the recombinant BirA in the fusion protein from the tag domain.
  • a preferred tag domain is derived from glutathione-S- transferase. That is it contains the active site of the glutathione-S- transferase enzyme.
  • a preferred binding reagent on the solid phase for use with such a tag domain is glutathione.
  • Other tag/binding reagent combinations known in the art are envisaged, such as polyhistidine tags, cellulose binding protein tags and S-tags (Novagen), plus suitable binding reagents.
  • Figure 1 shows a BirA protein encoding sequence suitable for use in the method according to the invention.
  • the database accession numbers for the BirA sequences from E. coli are (SwissProt) PO6709 and (Embl) g145430.
  • a similar sequence encoding the BirA protein containing conservative alterations that do not alter the protein sequence, or that do not significantly affect the biotinylation activity of the enzyme may be used.
  • a preferred plasmid expression vector contains a BirA encoding sequence fused to a glutathione-S-transferase tag, with a protease cleavable site positioned between the BirA sequence and the glutathione-S-transferase sequence.
  • a vector is preferably derived from one of the 'pGEX' vectors from Amersham-Pharmacia Biotec, though other purification tag vectors, known in the art are envisaged.
  • the recombinant BirA enzyme still comprising part of the fusion protein, is removed from the solid phase by means of one or more compounds capable of disrupting the binding of the tag domain to the binding reagent.
  • glutathione can be used where a glutathione-S-transferase tag and glutathione derivatised solid phase beads are used in the method.
  • the cleaving enzyme may have a tag domain similar to the tag domain of the fusion protein, which is capable of binding to the solid phase. This provides for the simple separation of the tag domain and the cleaving enzyme from the recombinant BirA enzyme by further passage across the solid phase.
  • An example of a commercially available cleaving enzyme possessing such a tag domain as the PreScissionTM protease (Pharmacia).
  • the BirA enzyme is purified after removal from the solid phase by one or more chromatographic steps to render the protein in a form suitable for use in biotinylation of peptides and proteins. This is particularly important where thrombin, or another protein cleaving substance, has been used to cleave the protein from all or part of the tag.
  • a preferred bacterial host strain is Escherichia coli , expression strain BL21 (DE3)pLysS.
  • Other bacterial strains amenable to the techniques of genetic manipulation, such as HMS 174, K-12, NovablueTM, AD494 and BLR and others known in the art, are also envisaged.
  • a preferred expression regulation sequence is the tac promoter, other promoters known in the art such as the T7 promoter may also be used.
  • the invention provides a bacterial expression vector containing an expression regulation sequence, operably linked to a sequence encoding a fusion protein of a BirA enzyme and a tag domain, which tag domain is suitable for separation of the fusion protein from a bacterial extract.
  • the fusion protein encoded by the vector contains a cleavage site to allow a functional BirA recombinant protein to be separated from part or all of the tag.
  • Preferred domains, expression regulation sequences and other elements of the vector are as discussed above.
  • the inventors have developed a rapid, simple and highly efficient method for the production and assay of the enzyme BirA.
  • an N-terminal GST tag By the addition of an N-terminal GST tag, production of the enzyme was enhanced by approximately 20 fold compared to previous reports.
  • N-terminal codon usage can significantly influence RNA secondary structure and the probability of successful translation.
  • the natural secondary structure of the 5' portion of the BirA mRNA favours a relatively low translation level compared to that of the GST portion of the fusion protein mRNA.
  • high level production of untagged BirA is not possible because of some negative influence of high levels of untagged BirA.
  • a repressor such as BirA
  • the addition of the tag may reduce the ability of the fusion protein to act as a repressor and so abolish or diminish any such effect.
  • purification was achieved in a straightforward fashion using the affinity of the recombinant protein for glutathione sepharose beads.
  • the free BirA could be readily released from the beads by incubation with thrombin, whilst keeping the protein at 4°C. All stages of the production and purification protocol are easily scaled up for commercial use. The bead purification was performed in batch mode and could be carried out on a very large scale after growth of bacteria in a fermenter.
  • the ion exchange step is easily scaled up.
  • the inventors have developed an assay which is cheap and highly suited for quality control of such a process to ensure appropriate levels of enzyme activity.
  • This protocol should prove to be the standard means for producing the BirA reagent. The cost effectiveness of this process should make it economically viable for commercial use.
  • Example 1 Protocol for the production of recombinant BirA protein
  • the gene encoding BirA was amplified by the polymerase chain reaction from DNA derived from Eschehchia coli using the primers C011 and C010 ( Figure 2) and cloned into the plasmid pGEX-2T (Amersham-Pharmacia Biotec, Uppsala, Sweden; GenBank Accession number U13850).
  • This plasmid contains a thrombin cleavage recognition sequence (Leu-Val-Pro-Arg-Gly-Ser [SEQ ID NO:2]) between a sequence encoding a glutathione-S-transferase tag and the cloning site into which he BirA cloning sequence is inserted.
  • the resulting plasmid, encoding BirA as a fusion protein with a glutathione-S- transferase tag was designated 'COC 053'.
  • the plasmid COC 053 was used to transform E. coli strain BL21 (DE3) pLysS (4). Transformed cells were grown on LB agar plates overnight with ampicillin selection (ampicillin present in the plates at 100 ⁇ g/ml).
  • the 1 litre culture was grown with aeration to mid log-phase growth (an optical density (Abs600 nm) of 0.6-0.8 AU), whereupon expression from the plasmid was induced with 0.4 mM isopropyl- ⁇ -D- thiogalactopyranoside (IPTG).
  • IPTG isopropyl- ⁇ -D- thiogalactopyranoside
  • Glutathione sepharose 4B beads (Pharmacia) were prepared by taking 1.33 ml of commercially available 75% slurry and spinning it at 500 x g in a 15ml Falcon tube for 5 minutes. The supernatant was removed and10 ml of cold PBS added before mixing. The mixture was centrifuged at 500 x g in a 15 ml Falcon tube for 5 minutes. The supernatant was removed and the pellet re-suspended with 1 ml of cold PBS to give a 50% slurry.
  • the sample was buffer exchanged into 20 mM Tris pH 8.4.
  • the PD 10 columns were pre-equilibrated with 30 ml of 20 mM Tris pH 8.4 and the supernatant applied to the column in a total volume of 2.5 ml, making up the volume to 2.5 ml with 20 mM Tris pH 8.4 if necessary.
  • the sample was eluted by applying a volume of 3.5 ml of 20mM Tris pH 8.4 to the gel bed.
  • Enzyme Assay Labelled reaction mixture 50mM Tris pH 7.4, 1 mM EDTA,
  • the scintillation count reflects the incorporation of labelled ATP into TCA precipitable material - i.e. the enzyme, which is protein. This process is biotin dependent. For this reason, there should be an obvious increase in the count between the biotin deficient and normal samples. Similarly, the more enzyme that is added, the higher the count; assuming that saturation has not occurred.
  • the inventors have provided a method that combines improved expression with an improved purification protocol which, in principle, could be scaled up to produce large amounts of protein not easily obtained by prior art methods.

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Abstract

L'invention concerne un procédé de production d'une enzyme BirA recombinante et des vecteurs d'expression codant ladite enzyme. Le procédé permet d'obtenir un rendement amélioré d'enzyme BirA pour une durée et un coût de purification de l'enzyme réduits.
PCT/GB1999/000245 1998-01-26 1999-01-25 PROTEINE BirA RECOMBINANTE Ceased WO1999037785A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GBGB9801621.5A GB9801621D0 (en) 1998-01-26 1998-01-26 Recombinant birA protein
GB9801621.5 1998-01-26

Publications (1)

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WO1999037785A1 true WO1999037785A1 (fr) 1999-07-29

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1295894A1 (fr) 2001-09-25 2003-03-26 F. Hoffmann-La Roche Ag Procédé pour une biotinylation spécifique en séquence de polypeptides in vitro
WO2002095013A3 (fr) * 2001-05-24 2004-01-15 Univ Technologies Int Biotine ligase mise au point par genie genetique pour biotinylation in vitro

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5252466A (en) * 1989-05-19 1993-10-12 Biotechnology Research And Development Corporation Fusion proteins having a site for in vivo post-translation modification and methods of making and purifying them
WO1995004069A1 (fr) * 1993-07-30 1995-02-09 Affymax Technologies N.V. Biotinylation de proteines

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5252466A (en) * 1989-05-19 1993-10-12 Biotechnology Research And Development Corporation Fusion proteins having a site for in vivo post-translation modification and methods of making and purifying them
WO1995004069A1 (fr) * 1993-07-30 1995-02-09 Affymax Technologies N.V. Biotinylation de proteines

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
ABBOTT J. AND BECKETT D.: "Cooperative binding of the Escherichia coli repressor of Biotin biosynthesis to the Biotin Operator sequence", BIOCHEMISTRY, vol. 32, 1993, pages 9649 - 9656, XP002106880 *
AUSUBEL F.M. ET AL.: "CURRENT PROTOCOLS IN MOLECULAR BIOLOGY: introduction to expression by fusion protein vectors; expression and purification of Glutathione-S-Transferase fusion proteins", 1994, WILEY & SONS, NEW YORK US, XP002106919 *
BUONCRISTIANI M.R. AND OTSUKA A.J.: "Overproduction and rapid purification of the Biotin Operon Repressor from Escherichia coli", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 263, no. 2, 15 January 1988 (1988-01-15), pages 1013 - 1016, XP002106879 *
KWEI-LAN TSAO ET AL.: "A versatile plasmid expression vector for the production of biotinylated proteins by site-specific, enzymatic modification in Escherichia coli", GENE, vol. 169, no. 1, 22 February 1996 (1996-02-22), pages 59-64, XP004042987 *
O'CALLAGHAN C.A. ET AL.: "BirA enzyme: production and application in the study of membrane receptor-ligand interactions bysite-specific biotinylation", ANALYTICAL BIOCHEMISTRY, vol. 266, no. 1, 1 January 1999 (1999-01-01), pages 9 - 15, XP002107534 *
SMITH D.B. ET AL.: "Single-step purification of polypeptides expressed in Escherichia coli as fusions with Glutathione S-Transferase", GENE, vol. 67, no. 1, 1 January 1988 (1988-01-01), pages 31 - 40, XP002033198 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002095013A3 (fr) * 2001-05-24 2004-01-15 Univ Technologies Int Biotine ligase mise au point par genie genetique pour biotinylation in vitro
EP1295894A1 (fr) 2001-09-25 2003-03-26 F. Hoffmann-La Roche Ag Procédé pour une biotinylation spécifique en séquence de polypeptides in vitro

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