WO1999037785A1

WO1999037785A1 - RECOMBINANT BirA PROTEIN

Info

Publication number: WO1999037785A1
Application number: PCT/GB1999/000245
Authority: WO
Inventors: Christopher Anthony O'callaghan; Bent Karsten Jakobsen
Original assignee: Isis Innovation Limited
Priority date: 1998-01-26
Filing date: 1999-01-25
Publication date: 1999-07-29
Also published as: GB9801621D0

Abstract

The invention relates to a method for the production of a recombinant BirA enzyme, and the expression vectors encoding the recombinant BirA enzyme. The method gives an improved yield of BirA enzyme and simultaneously decreases the time and cost of purification of the enzyme.

Description

Recombinant BirA Protein

This invention relates to a method for the production of recombinant BirA protein and to a vector encoding the protein.

Introduction

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

MDIRKIKKLIELVEESGISELEISEGEESVRISRAAPAASFPVMQQAYAAPM MQQPAQSNAAAPATVPSMEAPAAAEISGHIVRSPMVGTFYRTPSPDAKA FIEVGQKVNVGDTLCIVEAMKMMNQIEADKSGTVKAILVESGQPVEFDEP LWIE. 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, or biotinylation, is useful for many purposes, for instance a biotinylated protein can be separated from other unlabelled proteins by binding it to streptavidin. Although it has previously been possible to biotinylate proteins in other ways, the methods used are non- specific and usually result in biotin molecules being randomly attached at numerous sites all over the protein. As a result, 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.

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. Thus 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. When 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. As previously stated, 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 choice of a glutathione-S-transferase fusion tag as part of an expression vector encoding BirA was primarily made in an attempt to improve the recovery of the moderate amount of protein expected to be produced. An unexpected effect was observed which was the high level expression of the BirA protein at a level of 15-20% of total cell protein, compared to previously obtained levels of 0.5-1% (2,3). The observed high-level expression has provided a much improved and convenient method for the production of milligram quantities of highly active recombinant BirA.

Summary of the Invention

Thus 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. Using the method according to the invention, it is possible to obtain expression of recombinant BirA enzyme in an E. coli expression system, to high level, such as 5% or more, or 10% or more, or 15% or more, of the total cellular protein by mass.

In a preferred embodiment of the method, the recombinant BirA enzyme is removed from the solid phase by cleaving the recombinant BirA enzyme of the fusion protein from the tag domain. Preferably 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 PreScission™ 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. Clearly, 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. Such 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.

In an alternative embodiment of the method of the invention, 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. For example glutathione can be used where a glutathione-S-transferase tag and glutathione derivatised solid phase beads are used in the method.

In this alternative embodiment, it is possible to remove the tag domain from the recombinant enzyme by cleavage with a suitable enzyme after elution from the solid phase. Optionally, 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 PreScission™ protease (Pharmacia).

Preferably, 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, Novablue™, 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.

In another aspect 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.

In a preferred embodiment 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.

Discussion

The inventors have developed a rapid, simple and highly efficient method for the production and assay of the enzyme BirA. By the addition of an N-terminal GST tag, production of the enzyme was enhanced by approximately 20 fold compared to previous reports. There are several possible reasons for this enhancement. It is well recognised in the art that N-terminal codon usage can significantly influence RNA secondary structure and the probability of successful translation. It is possible that 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. In addition, it is possible that high level production of untagged BirA is not possible because of some negative influence of high levels of untagged BirA. In particular, it is possible that the presence of an excess of a repressor such as BirA could be deleterious to cell growth. 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. With such a high level of primary expression, purification was achieved in a straightforward fashion using the affinity of the recombinant protein for glutathione sepharose beads. In addition, 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. Similarly, the ion exchange step is easily scaled up. In addition, 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.

The invention will now be further described in the Examples which follow, which are not intended to limit the scope of the invention in any way.

EXAMPLE

Example 1 : Protocol for the production of recombinant BirA protein

Plasmid construction

Using methods well known in the art, 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'.

Bacterial growth

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 μg/ml).

All growth was at 37°C. A single colony from a plate of transformed E. coli was used to inoculate 2 ml of Typ medium (16g bactotryptone, 16g bacto yeast extract, 5g sodium chloride, 2.5g potassium dihydrogen phosphate per litre) with ampicillin present at 100 μg/ml which was established for 3 hours with aeration.

This was inoculated into 250ml of Luria Broth medium, made with 10 g bacto-tryptone, 5 g yeast extract and 5 g sodium chloride and supplemented with ampicillin at 100 μg/ml and left to stand overnight. The next day 25ml aliquots of this culture were inoculated into 1 litre flasks.

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-β-D- thiogalactopyranoside (IPTG). Cells were harvested after 4 hours further growth by centrifugation at 3000 rpm in a Beckman J-6B swinging bucket centrifuge. The supernatant was discarded and the cell pellet retained.

Cell lysis and clarification The cell pellet, either immediately or after storage at -80 °C, was re-suspended in cold phosphate buffered saline (PBS) and sonicated to lyse the cells. Adequate sonication is critical and it is important to make sure that the preparation remains cool. For this reason, a large re- suspension volume of around 100 ml is helpful and if possible the procedure should be carried out in a glass container in an ice and water bath or the preparation repeatedly returned to cool on ice in between 20 second bursts of sonication. The volumes given below assume that the preparation is lysed in 100 ml. After sonication, add 5 ml of 20% Triton X- 100 (Sigma) to give a final concentration of 1 % and leave this mixing at 4°C for 30 minutes, preferably with gentle agitation. Centrifuge the entire preparation at 12000 x g for 30 minutes at 4 °C. Remove and retain supernatant. Discard the pellet.

Purification Bead preparation

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.

Affinity purification

Affinity purifcation of the BirA-glutathione-S-transferase fusion protein was carried out by the following method:

• Add the 2 ml of 50% slurry to the cell supernatant.

• Gently agitate at 4 °C for 30 minutes.

• Centrifuge the mixture of beads and cell supernatant at 500 x g for 5 minutes and remove and discard the supernatant.

• Add 20 ml of PBS and re-suspend the beads. Centrifuge at 500 x g in a 15 ml Falcon tube for 5 minutes. Remove supernatant.

• Add 20mls of PBS and re-suspend the beads. Centrifuge at 500 x g in a 15 ml Falcon tube for 5 minutes. Remove supernatant.

Thrombin cleavage of protein

To separate the glutathione-S-transferase tag from the recombinant BirA protein the following protocol was followed: Make thrombin (Sigma Chemicals) stock solution up at 1000 cleavage units per ml in PBS. Add 20 μl of thrombin stock solution to 1 ml of PBS and add this to the beads. Incubate for 12 hours at 4 °C with agitation. Spin at 500 x g for 5 minutes and remove the supernatant. Run a sample of the supernatant on a 15 % SDS-polyacrylamide gel. A band of approximately 30 kDa should be seen.

Regeneration of beads

To regenerate the beads, 10 ml of fresh glutathione elution buffer (10 mM reduced glutathione, 50 mM Tris pH 8.0) was used to re- suspend the beads, which were then left at 4 °C for 2 hours. The beads were then centrifuged at 500 x g for 5 minutes the supernatant discarded. This elution was repeated a further two times and the beads finally stored by adding 5 ml of PBS and keeping at 4 °C.

Chromatographic purification.

Buffer exchange.

The sample was buffer exchanged into 20 mM Tris pH 8.4.

This can be performed using disposable PD10 columns from Pharmacia.

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.

Ion exchange chromatographv Preparative chromatography was performed on a Pharmacia

FPLC chromatography system using a Mono Q HR 5/5 (1 ml) column. The preparation was centrifuged at 12,000 x g for 5 min and filtered through a 0.22 μm filter. The following buffers, also filtered to 0.22 μm, were used: Buffer A (20 mM Tris pH 8.4); Buffer B (20 mM Tris pH 8.4, 1 M NaCl). The Mono Q column was prepared by running 10 ml of Buffer

A, followed by 10 ml of Buffer B, followed by a further 30mls of Buffer B until the baseline absorbance at 280 nm was flat. A flow rate of 0.5 ml per minute is reasonable. The column was then equilibrated with 10 ml of Buffer A prior to sample application. The BirA / thrombin sample was applied to the column and the column washed with 10 ml of Buffer A. A 30 ml gradient from 0 to 50% Buffer B was then run through the column. A single peak corresponding to BirA eluted at approximately 16% in the gradient (160 mM NaCl). The concentration of enzyme was measured (usually around 0.5 mg/ml) and an assay of enzymatic activity performed. Once this purification procedure has been carried out the enzyme can be stored for at least 9 months at -80°C with routine freezing, showing negligible activity loss.

Enzyme Assay Labelled reaction mixture (50mM Tris pH 7.4, 1 mM EDTA,

0.75M KCI, 10mM Mg acetate, 1 mM DTT (dithiothreitol), 1mM unlabelled ATP (adenosine triphosphate), 1 μCi tritiated ATP (Amersham, UK) 8mM biotin, 0.2 units of inorganic pyrophosphatase (reagents from Sigma, UK unless otherwise specified) were established in triplicate with different concentrations of BirA enzyme in 1 ml volumes in Eppendorf tubes at room temperature. Controls were also established with no biotin or no ATP or no BirA enzyme. Reactions were incubated for two hours and then 25μl aliquots from each reaction dispensed with a positive displacement pipette onto labelled squares of paper (Whatman, UK). The paper squares were then washed twice in ice cold tricarboxylic acid with stirring on ice for 5 minutes, then in water for 5 minutes, then ethanol for 5 minutes and finally ether for 5 minutes. The paper was then allowed to dry in a fume cupboard and each square placed in a scintillant bottle with 10mls of non aqueous scintillant fluid and counted with a Beckman scintillation counter.

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.

Results

The addition of an N-terminal GST tag to the protein resulted in markedly enhanced expression compared to that of the untagged protein in our own experience (results not shown) and in the literature. The recombinant protein accounted for around 15-20% of total cellular protein. The use of the GST tag in conjunction with glutathione sepharose beads provided a highly efficient means of purifying soluble, recombinant protein away from other cellular protein. Solid phase cleavage of the recombinant protein, whilst it was bound to the beads, liberated the free BirA moiety of the fusion protein in an active state. Thrombin was readily separated from BirA using anion exchange at pH8.4. The enzyme was stored in the ion exchange effluent buffer at -80°C and was stable for months post- production.

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.

References

1. Altman JD et al. (1996) Phenotypic analysis of antigen- specific T-lymphocytes. Science 274:94-96

2. Buoncristiani M and Otsuka AJ (1988) Overproduction and rapid purification of the biotin operon repressor from Eschehchia coli. J. Biol. Chem. 263:1013-1016

3. Abbot J and Beckett D (1993) Cooperative binding of the

Eschehchia coli repressor of biotin biosynthesis in the biotin operator sequence. Biochemistry 32:9649-9656

4. Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW (1990) Use of T7 RNA-polymerase to direct expression of cloned genes Meth. Enzymol. 185:60-89

Claims

1. 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.

2. A method according to claim 1 wherein the recombinant BirA enzyme is removed from the solid phase by cleaving the recombinant BirA enzyme of the fusion protein from the tag domain.

3. A method according to claim 2, wherein cleavage is by enzymatic means.

4. A method according to any one of claims 1 to 3, wherein the tag domain is derived from glutathione-S-transferase,

5. A method according to claim 4, wherein the binding reagent is glutathione.

6. A method according to any one of claims 1 to 5 wherein the sequence encoding the fusion protein comprises the BirA protein encoding sequence of figure 1.

7. A method according to claim 1 wherein 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.

8. A method according to any one of claims 1 to 7, wherein 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.

9. A method according to any one of claims 1 to 8, wherein the bacterial host strain is Eschehchia coli BL21 (DE3) plysS.

10. A method according to any one of claims 1 to 9, wherein the expression regulation sequence is the tac promoter.

11. 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.

12. A vector according to claim 11 wherein the tag domain is derived from glutathione-S-transferase.

13. A vector according to claim 11 or claim 12, wherein the expression regulation sequence is the tac promoter.

14. A vector according to any one of claims 11 to 13, wherein the fusion protein contains a cleavage site to allow a functional BirA recombinant protein to be separated from part or all of the tag.

15. A vector according to claim 14 wherein the cleavage site is cleavable by thrombin.