WO2015165841A1 - An antibiotic-free method for selection of transformed bacteria - Google Patents

Abstract

An antibiotic-free selection method, which is based on the complementation of hemA deficient bacteria by a vector carrying a functional hemA gene, as a selection marker. The hemA gene encodes an active glutamyl-tRNA reductase, the expression of which is required for bacterial growth in the absence of sufficient 5-ALA and hemin. The hem A gene of the vector can therefore be used as selection marker for plasmid maintenance in hem A deficient bacteria.

Description

AN ANTIBIOTIC-FREE METHOD FOR SELECTION OF TRANSFORMED

BACTERIA

FIELD OF THE INVENTION

[1] The present invention relates to antibiotic-free selection of transformed bacteria for recombinant protein production and/or propagation of vector(s).

BACKGROUND ART

[2] Essential requirements for succeeding in heterologous protein expression or plasmid DNA preparation using genetically engineered bacteria are the ability to efficiently uptake and maintain a vector codifying for a particular gene of interest. Most commonly a selection marker is included in the vector. The selection marker can be a gene or DNA sequence that allows separation of the bacteria containing the marker and those not containing it. The combination of the selection marker and the selection medium can allow for the growth of bacteria that have been transformed with the vector, while prohibiting the growth of bacteria that have not been transformed.

[3] Antibiotic resistance genes are the most commonly used markers for selecting and maintaining recombinant plasmids in hosts, such as Escherichia coli. An antibiotic resistance gene as a selection marker, in combination with a medium containing the antibiotic, can be used in order to achieve selection. However, the use of antibiotic resistance genes contributes to the problem of widespread antibiotic resistance (Levy S.B., 2002). In addition the increasing regulatory requirements to which biological agents are subjected will have a great impact in the field of industrial protein expression and production. There is therefore an increasing need for improved antibiotic-free selection systems.

[4] Previously disclosed antibiotic-free selection systems include the use of auxotrophic strains, for instance, amino acid auxotrophic E.coli strains, that can be used in combination with genes, that overcome the specific auxotrophy, as selection markers. These methods are able to effectively restore the growth of a mutant strain in a defined medium, i.e. one that lacks the specific nutrient that is required for growth of the auxotrophic mutant strain. However, such selection methods do not consistently provide stringent selection against the growth of untransformed bacteria. Further, since many culture media used are complex and contain amino acids that would permit growth of the mutant strains, it is necessary to use chemically defined media that do not contain the relevant amino acid.

SUMMARY OF THE INVENTION

[5] The present invention provides a microbial expression system for the production of polypeptides based on the use of extrachromosomal DNA, whereby no antibiotic marker genes (genes whose derived proteins provide the cell with resistance to an antibiotic, also named antibiotic-resistance genes, resistance genes, antibiotic maker or antibiotic selection marker) for the selection of the host cell but DNA sequences that code glutamyl tRNA-reductase (also referred to as hemA genes) are used such that the production of the desired polypeptide, e.g., vaccine antigens and CRM197, does not need the addition of antibiotics. The expression system is free from antibiotic-resistance genes. An advantage of this approach over other auxotrophic strains is that 5-aminolevulinc acid (5 -ALA), which can be used to restore the growth of heniA- defective strains, is not present in complex media and therefore such media (e.g. yeast extract, tryptone), which are often used in bacterial growth media, can still be used. We also found that using approach, a high plasmid copy number was maintained, as compared with conventional antibiotic resistance-based selectable marker systems.

[6] Thus, in a first aspect, the invention provides a method for antibiotic-free selection of transformed bacteria comprising the steps of (i) transforming a bacterial cell lacking a functional hemA gene with a vector comprising a first nucleic acid sequence encoding a functional glutamyl tRNA-reductase and a second nucleic acid sequence encoding a protein of interest to give a transformed bacterial cell and (ii) growing the transformed bacterial cell in a growth medium that does not support growth of bacterial cells lacking a functional hemA gene in the absence of the vector. Particularly the vector is an expression vector and yet more particularly the vector is a non-integrative vector. The term "non-integrative" as used herein in reference to a vector indicates that the vector does not integrate into the host DNA, i.e. it does not integrate into the bacterial chromosome, and replicates extra-chromosomally.

[7] As used herein, the term "antibiotic-free selection" refers to the use of conditions that enable the discrimination of cells displaying a required phenotype without the use of antibiotics, e.g., the growth of bacteria in medium wherein antibiotic(s) are not used to select for cells containing a nucleic acid of interest. [8] Particularly the growth medium will comprise no more than, or less than, 2.5mg/L of 5- aminolevulinc acid (5-ALA) and when the bacteria are heme permeable, i.e. they carry one or more genes encoding a hemin uptake protein (hem-p), the growth medium will comprise no more than, and preferably less than, 4mg/L of hemin. Particularly the growth medium is an undefined, complex medium, for example, LB broth although defined medium may also be used.

[9] In some embodiments, the bacterial cell will comprise a mutation in the hemA gene which reduced or abolishes glutamyl tRNA-reductase activity. Particular point mutations numbered in accordance with SEQ ID NO:2 include, by way of non- limiting example, G7D, C50S, R52Q, G106N, E114K, S145F, G191D, R314C and combinations thereof. Further combinations may include G44C/S105N/A326T and S22L/S164F. In some embodiments the bacterial cell is an Escherichia coli strain such as SASX41B (CGSC#: 4806), HU227 (CGSC#: 8002) and EV61.

[10] In other embodiments, the bacterial cell is a bacterial strain is a hemA knock-out. The term "knock-out" as used herein means cells comprise a loss-of-function modification of the hemA gene the end result of which is that no protein with the normal function of the wild-type gene product is made from the gene or only a protein with diminished function is made. Particularly the cell is a strain of Escherichia coli (E.coli) selected from the group consisting of BL21(DE3) AhemA or DH5alpha AhemA and HK100(DE3) AhemA wherein AhemA refers to deletion of the hemA gene.

[11] In a second aspect of the invention, there is provided a non-integrative expression vector comprising (i) as a selectable marker, a first nucleic acid sequence encoding a functional glutamyl tRNA-reductase, operably linked to a first regulatory control sequence that directs expression of the glutamyl tRNA-reductase in a bacterial host cell; (ii) a multiple cloning site (MCS) and (iii) a second regulatory control sequence operably linked to the MCS such that a second nucleic acid of interest inserted into the MCS will be expressed, separately from the glutamyl tRNA-reductase, in a bacterial host cell comprising the expression vector. The expression vector does not encode a polypeptide involved in tetrapyrrole synthesis other than glutamyl tRNA-reductase (i.e. the second nucleic acid sequence of interest, when present, does not encode a polypeptide involved in tetrapyrrole synthesis).

[12] In one embodiment, the expression vector further comprises a second nucleic acid sequence encoding a protein of interest inserted into the MCS. Thus the present invention also provides a non-integrative expression vector for complementing a bacterial cell lacking a functional hemA gene the vector comprising a first nucleic acid sequence encoding a functional glutamyl tRNA-reductase and a second nucleic acid sequence encoding a protein of interest.

[13] The protein of interest may for example be a therapeutic polypeptide and/or immunogenic.

[14] An active glutamyl tRNA-reductase is expressed from said first nucleic acid: typically the first nucleic acid is a hemA gene. The first nucleic acid sequence encoding a functional glutamyl tRNA-reductase is operably linked to a first regulatory control sequence, such as a promoter, for example, the P3 promoter from the ampC gene.

[15] The second nucleic acid sequence may be operably linked to either the first promoter or a second promoter. In some embodiments the second promoter may be the same as the first promoter. In other embodiments this promoter may be a different promoter, particularly an inducible promoter such as, by way of non-limiting example, an araB, hsp70 or LacZYA promoter. Those skilled in the art will be aware of other suitable promoters.

[16] Generally the vector will comprise an origin of replication, for example the Fl or ColEl origins of replication. Generally the vector will comprise a multiple cloning site (MCS) - a region containing one or more unique restriction endonuclease recognition sites to enable a nucleic acid sequence to be cloned into the vector. In particular embodiments, the second nucleic acid sequence encoding a protein of interest is inserted into the MCS. The expression vector may be derived from a pET24 plasmid although the skilled person will understand that other plasmid backbones may be utilised.

[17] In a third aspect of the invention, there is provided the use of the vector of the second aspect in the method of selection of the first aspect as well as in a method of producing a protein of interest.

[18] In a fourth aspect of the invention, there is provided a bacterial cell lacking a functional hemA gene which contains an expression vector of the second aspect, wherein the functional glutamyl tRNA-reductase of the expression vector complements hemA deficiency in the bacterium. [19] In a fifth aspect of the invention there is provided a method of producing a protein of interest, comprising a step of growing a bacterial cell lacking a functional hemA gene under conditions such that (a) the growth of a bacterial cell lacking a functional hemA gene is not supported in the absence of a complementary hemA gene, and (b) the protein of interest is produced. Typically the method also comprises the step of (c) purifying the protein of interest from the cell(s) and/or growth medium. In certain embodiments there is provided the use of hemA deficient bacteria in a method of selection of transformed bacteria. A further step of the method comprises preparing a pharmaceutical or vaccine composition comprising the purified protein.

[20] In a related aspect the present invention provides a method of producing a protein of interest which method comprises the steps of: i) providing a bacterial cell comprising (a) a non-integrative vector comprising a first nucleic acid sequence encoding a functional glutamyl tR A-reductase operably linked to a first regulatory control sequence that directs expression of the glutamyl tRNA-reductase in the bacterial host cell and (b) a second nucleic acid sequence encoding the protein of interest operably linked to a second regulatory control sequence that directs expression of the protein of interest in the bacterial host cell; ii) growing the host cell under conditions that do not support growth of bacterial cells that lack a functional glutamyl tRNA-reductase; and optionally iii) recovering the protein of interest from the host cell or culture medium, wherein the protein of interest is not a protein involved in tetrapyrrole synthesis.

[21] In a sixth aspect of the invention, there is provided a kit containing a bacterial cell lacking a functional hemA gene and an expression vector of the second aspect of the invention. Particularly the kit is for use in a method of the first or fifth aspects.

BRIEF DESCRIPTION OF THE DRAWINGS

[22] Figure 1 : shows a plasmid map of the pHem plasmid of SEQ ID NO: 8 and SEQ ID NO:9. [23] Figure 2a: shows the growth of the hemA/HU227 mutant with and without the pHem- BFP plasmid in defined medium (M9+glucose). The graph shows OD600nm against time (0-10 hours). HemA/HU227 is represented by squares and HemA/HU227 + pHem-BFP is represented by circles.

[24] Figure 2b: shows the growth of the hemA/HU227 mutant with and without the pHem- BFP plasmid in complex medium (LB). The graph shows OD600nm against time (0-10 hours). HemA/HU227 is represented by squares and HemA/HU227 + pHem-BFP is represented by circles.

[25] Figure 3a: shows the growth of four amino acid auxotrophy mutants, with a complementation plasmid or with the auxotrophic amino acid, in defined media. The graph shows OD600nm against time (0-10 hours). Supplementation of the amino acid was at a final concentration of: L-arginine and L-proline 100 mg/L; L-lysine 30 mg/L; and L-cysteine 0.4mM. The growth of the auxotrophic strain is represented by the upper line, the growth of the auxotrophic strain supplemented by the auxotrophic amino acid is represented by middle line, and the growth of the auxotrophic strain complemented by a plasmid is represented by the lower line.

[26] Figure 3b: shows the growth of four amino acid auxotrophy mutants, with a complementation plasmid or with the auxotrophic amino acid, in complex media. The graph shows OD600nm against time (0-10 hours). Supplementation of the amino acid was at a final concentration of: L-arginine and L-proline 100 mg/L; L-lysine 30 mg/L; and L-cysteine 0,4mM. The growth of the auxotrophic strain is represented by crosses, the growth of the auxotrophic strain supplemented by the auxotrophic amino acid is represented by squares, and the growth of the auxotrophic strain complemented by a plasmid is represented by circles.

[27] Figure 4: shows recombinant protein production in the 5 -ALA auxotrophy selection method, in comparison to the conventional antibiotic selection method. The graph shows the specific production fluorescence/O. D (a.u.) for "NI", the negative control where expression was not induced, and "I" where expression was induced. In each pair of bars, BL21(DE3)hemA::kan + pHem-BFP (no antibiotics) is shown on the left, and BL21(DE3) +pHem-BFP (Kanamycin supplemented) is shown on the right for each of "NI and "I". [28] Figure 5: shows specific fluorescence/OD (a.u.) for BFP production in the transformed bacteria after 15 cultivation cycles. For each number of cycles, hemA- bacteria are shown on the left and BL21 bacteria are shown on the right.

[29] Figure 6: shows the role of hemA in the 5 -ALA biosynthesis pathway.

[30] Figure 7a: shows the comparative growth rates (optical density LogOD600nm) against time(h) of BL21(DE3) for (i) Ahem/pHem (ii) pET24/wt and (iii) pET24/wt +K. Data reported are average of three biological replicas.

[31] Figure 7b: shows the comparative growth rates (optical density LogOD600nm) against time(h) of pHem/DH5 Ahem and pET24/ DH5 (wild type). Data reported are average of three biological replicas.

[32] Figure 7c: shows the comparative growth rates (optical density LogOD600nm) against time(h) of pHem/HK100(DE3) Ahem and pET24/HK100(DE3) (wild type). Data reported are average of three biological replicas.

[33] Figure 8: shows the recombinant GFP expression (specific fluorescence RF/OD a.u.) of (i) pET24-GFP/BL21(DE3) (left), (ii) pET24-GFP/BL21(DE3) with kanamycin (middle) and (iii) pHem-GFP/BL21 (DE3)AhemA (right) at 1, 5, 10, 15 and 20 passages of the E.coli.

[34] Figure 9: shows BL21(DE3)AhemA growth rate (represented by OD600nm) at varying 5- ALA concentrations against time(h).

DETAILED DESCRIPTION

[35] Antibiotic selection methods and methods relying on auxotrophic selection require the addition of one or more additives to media. As a result they can be more expensive, particularly if the additive must be supplied during the whole cultivation. Widespread use of antibiotics may contribute to the spreading of resistance genes to pathogenic bacteria which might have negative consequences on disease control. Industrially used fermentation media usually contain components that are waste products of other, often fermentative processes, e.g., grain residues from the ethanol production. Thus, fermentation media are generally very complex and it is difficult to maintain a selection pressure in these media when auxotrophic strains are used. In auxotrophic bacteria, some gene function may remain such the selection is not tightly controlled and is leaky. Therefore, there is a need for improved methods of bacterial selection that are antibiotic-free and wherein the selective pressure can occur in both complex and defined media. Ideally the selection method will provide a high plasmid copy number and a high amount of recombinant protein production with respect to antibiotic-based selection methods.

[36] The inventors have found that the use of a hemA gene in a method for the selection of bacteria satisfies this need. In particular the hemA system developed by the inventors is tightly regulated, non-leaky and does not require the inclusion of additives to growth media. In contrast to selection systems known in the art, the growth profile of bacteria in the present invention also more closely mimics those of wild-type bacteria.

[37] The hemA gene encodes an active glutamyl-tR A reductase, the expression of which is required for bacterial growth. Figure 6 shows the role of hemA in the 5-aminolevulinc acid (5- ALA) biosynthesis pathway. HemA deficiency prevents synthesis of 5-ALA (Chen et al., 1994). 5 -ALA is the first compound in the porphyrin synthesis pathway that leads to Heme, which is required for respiration metabolism in bacteria since it is the prosthetic group of cytochromes.

[38] The present invention is based on antibiotic-free plasmid selection based on complementation of hemA deficient bacteria by a vector carrying a functional hemA gene, as a selection marker, which confers to the transformed bacteria the ability to grow and maintain the vector in any medium that does not support growth of hemA deficient bacteria.

[39] The method for selection of transformed bacteria comprises transforming hemA deficient bacteria with a vector carrying a functional hemA gene, as a selection marker, to give transformed bacteria; and growing the transformed bacteria in a growth medium that does not support growth of hemA deficient bacteria in the absence of a vector carrying a functional hemA gene. The expression vector for complementing the hemA deficient bacteria, carries a functional hemA gene as a selection marker from which an active glutamyl tR A-reductase is expressed.

[40] In contrast to amino acid based auxotrophy systems, the use of the hemA gene allows for selection to occur in complex growth media. In amino acid auxotrophy based selection methods, such as those tested in example 1 , the untransformed mutant amino acid auxotrophic strains are able to grow in complex media, such that selection based on the restoration of growth by the vector is not possible in complex media. As demonstrated in example 1, the absence of significant amount of 5-ALA in the components used in formulation of known complex media means that the hemA-based selection method of the present invention is not restricted to chemically defined growth media. Chemically defined growth media generally provide lower yields and are more expensive than complex media, especially if the list of media components includes growth factors and vitamins. Therefore the development of an expression system that guarantees reproducible results in both complex media and chemically defined growth media is strongly desirable and advantageous.

[41] Surprisingly, the system of the present invention exhibits a higher plasmid copy number than conventional antibiotic based systems, as demonstrated in example 2. It has also been demonstrated that hemA deficient bacteria of the invention, when complemented with a hemA expressing plasmid and cultivated in standard conditions and the absence of antibiotic, produce more recombinant protein than wild type bacteria that have been transformed with the plasmid and grown in the same conditions in the presence of antibiotic.

[42] This increase in recombinant protein production has also been shown to be maintained over many generations, and 20 cultivation cycles. Example 4 shows that whereas production of recombinant protein from the antibiotic based system significantly decreases as the number of cultivation cycles increases, higher recombinant protein production is maintained for longer in the hemA based selection method. Furthermore, the examples show that the enhanced recombinant protein production is not limited to any particular protein. Example 4 shows enhanced recombinant protein production for BFP and GFP, and example 6 shows enhanced recombinant protein expression for two antigens, STA011 and GBS 1523-80.

HemA:

[43] Complementation of hemA deficient bacteria with an expression vector comprising a functional hemA gene allows transformed bacteria to grow in the absence of 5-ALA and/or hemin. Selection occurs when the bacteria are grown in a media that will support the growth of the transformed bacteria, but not the untransformed bacteria.

[44] The functional hemA gene comprises any DNA sequence that is capable of expressing an active glutamyl-tRNA reductase. An active glutamyl-tRNA reductase is capable of converting L- glutamyl-tRNA into glutamate-l-semialdehyde. The hemA gene particularly comprises the nucleic acid sequence identified in SEQ ID NO: 1, and particularly encodes the active glutamyl- tRNA reductase identified in SEQ ID NO:2: [45] MTLLALGINHKTAPVSLRERVSFSPDKLDQALDSLLAQPMVQGGVVLSTCNRTELYLSV

EEQDNLQEALIRWLCDYHNLNEEDLRKSLYWHQDNDAVSHLMRVASGLDSLVLGEPQILGQVKK AFADSQKGHMKASELERMFQKSFSVAKRVRTETDIGASAVSVAFAACTLARQIFESLSTVTVLL VGAGETIELVARHLREHKVQKMI IANRTRERAQILADEVGAEVIALSDIDERLREADI I I SSTA SPLP I IGKGMVERALKSRRNQPMLLVDIAVPRDVEPEVGKLANAYLYSVDDLQS I I SHNLAQRK AAAVEAETIVAQETSEFMAWLRAQSASETIREYRSQAEQVRDELTAKALAALEQGGDAQAIMQD LAWKLTNRLIHAPTKSLQQAARDGDNERL ILRDSLGLE

[46] The hemA gene preferably encodes a protein with a sequence identity that is greater than 50% (e.g. 60%, 70%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%) or more) with respect to SEQ ID NO: 2. Typically, 50%> identity or more between two proteins is considered to be an indication of functional equivalence. Identity between proteins is preferably determined by the Smith Waterman homology search algorithm as implemented in the MPSRCH program (Oxford Molecular), using an affine gap search with parameters gap open penalty=12 and gap extension penalty=2. Exemplary protein sequences from E. coli include GenBank Accession Nos: AAC74294.1 (GI: 1787461, glutamyl-tRNA reductase) and AAA23954 (GI: 146333, delta-aminolevulinic synthase). Such proteins may be encoded by DNA sequences including Genbank Accession No: M25323.1 (GL 146331, Escherichia coli delta-aminolevulinic synthase (hemA) gene, complete cds). HemA genes are also found in other species of bacteria, including Staphylococcus aureus (EWM96411.1 GL585885121) SEQ ID 3:

[47] MHFIAI S INHRTADVALREQVAFRDDALRIAHEDLYETKS ILENVILSTCNRTEVYAVV DQIHTGRYYIQRFLARAFGFEVDDIKAMSEVKVGDEAVEHLLRVTSGLDS IVLGETQILGQIRD AFFLAQSTGTTGTIFNHLFKQAITFAKRVHNETDIADNAVSVSYAAVELAKKVFGKLKSKQAII IGAGEMSELSLLNLLGSGITDITVVNRTIENAMKLAAKHQVKYDELSSLPNLLESADIVISSTS AQSYI ITNEMIERIAENRKQDSLVLIDIAVPRDIEPGI SAITNIFNYDVDDLKGLVDANLRERQ LAAATI SEQIPAEIHAHNEWI SMLGVVPVIRALREKAMAIQAETMDS IDRKLPGLSERERKI I S KHTKS I INQMLKDP IKQAKELSSDKKSNEKLELFQ IFDIEAECPHEQAKQQKESKVKEI SARR IFSFE ,

Pseudomonas aeragmosa,,(WP_003125353.1 GL489216779) SEQ ID 4:

[48] MAFIALGINHKTASVAVRERVAFTPEQMVEALQQLCRLTTSREAAILSTCNRSELYLEI DHPTADDVLAWLADYHRLTLDELRACAYVHQDEDAVRHMMRVASGLDSMVLGEPQILGQMKSAY AVAREAGTVGPLLGRLFQATFSTAKTVRTDTAIGENPVSVAFAAVSLAKQIFSDLHRSQALLIG AGETITLVARHLFEQGVKRIVVANRTRERASLLAEQFGAHAVLLSEIPEELANSDIVISSTASQ LP ILGKGAVERALKQRKHKPMFMVDIAVPRDIEPEVGELDDVYLYSVDDLHEVVAENLKSRQGA AQAAEELVGSGVAEFMQRLRELAAVDVLRAYRQQAERLRDEELGKAQRQLANGADPAEVMAQLA RGLTNKLLHAPSVQMKKMSAEGRIDALALAQELFALDEGAPRH,

Bacillus halodurans (NP 243914.1 GL 15615610) SEQ ID 5:

[49] MHILMIGLNYKTAPVEIREKFSFQDSELPQALHQLRQMKS ILECTIVSTCNRTELYVVA DQLHTGRHFTKTFLADWFKLPKDEFTPFLTIRENDHAIEHLFRVVTGLDSMILGETQILGQVRN SFFIAQEEQVTGS IFNHLFKQAITLAKRAHSETDIGQNAVSVSYAAVELGKKIFDDFKGKQVLI LGAGKMGELTAKHLHSNGAEQVTVINRTREKAAELAKRFLGVDRPYNELTEAIVEADILI SSTG ATGYVVTSDMVSHALKKRKGRPLFMVDIAVPRDLDPALASHDDVYLYDIDDLQNIVQTNLEERR TEAEKIELLIEEELVEFKQWLNTLGVVP I ITALRTKALTVQGETMES IERKLPNLTEREKKVLR KHTKS IVNQLLRDP ITRIKELANAPEREEALDLFTKIFALEEELAEQEKQEKVKQAEQEWLAKK RP ITCMEKQSHVMVKS , and Salmonella enterica (WP 023260590.1 GL555285828) SEQ ID 6:

[50] MTLLALGINHKTAPVSLRERVTFSPDTLDQALDSLLAQPMVQGGVVLSTCNRTELYLSV EEQDNLQETLIRWLCDYHNLNEDDLRNSLYWHQDNDAVSHLMRVASGLDSLVLGEPQILGQVKK AFADSQKGHLNASALERMFQKSFSVAKRVRTETDIGASAVSVAFAACTLARQIFESLSTVTVLL VGAGETIELVARHLREHKVQKMI IANRTRERAQALADEVGAEVI SLSDIDARLQDADI I I SSTA SPLP I IGKGMVERALKSRRNQPMLLVDIAVPRDVEPEVGKLANAYLYSVDDLQS I I SHNLAQRQ AAAVEAETIVEQETSEFMAWLRAQGASETIREYRSQSEQIRDELTTKALSALQQGGDAQAILQD LAWKLTNRLIHAPTKSLQQAARDGDDERLNILRDSLGLE

[51] Such hemA genes are also suitable for use as selection markers of the invention. Those of skill in the art can readily identify native coding sequences of the above exemplary bacterial proteins or determine nucleic acid sequences that would encode the protein sequences above. Preferably, the hemA gene used as a selection marker will be from the same species as the hemA deficient bacteria that is subject to complementation. Thus the hemA gene which complements a hemA deficiency may be identical to the hemA gene from the parental wild-type strain, for example, the wild-type strain from which a hemA deletion mutant strain is derived. However, in certain embodiments, the hemA gene used as a selection marker for complementation will be different from the species of the hemA deficient bacteria. [52] Active glutamyl-reductases encoded by the hemA gene can take various forms, including fusion proteins. The functional hemA gene can also comprise a fragment of a hemA gene or a hemA gene containing one or more internal deletions or mutations within the hemA gene which encodes an active glutamyl-tR A reductase. As used herein, an "active glutamyl-tR A reductase" is a glutamyl-tRNA reductase that when expressed under the control of a suitable promoter, such as P3, in a suitable AhemA cell line, such as the K12 derived AhemA HU227 cell line provided herein, permits significant growth (p < 0.05) of the cells in LB media within an 8 hour time period as determined by at optical density measurement at 600 nm (OD600).

HemA deficient bacteria:

[53] The invention provides for the use of HemA deficient bacteria in the method of selection of transformed bacteria of the present invention. The term "hemA deficient" and "bacterial cell lacking a functional hemA gene" are used interchangeably to refer to expression of the gene in the cell. In some embodiments, the cell is null for the hemA gene in that it has no copies of the gene and is, therefore unable to express the gene. In some embodiments, the status of the gene or gene product is that it is mutated such that the gene is not expressed or that the gene product is not functional or has significantly less function than the wild-type gene. Accordingly, a cell that is deficient in the expression of the hemA gene may have no hemA gene or the hemA gene may be mutated so that the hemA gene product, glutamyl-tRNA reductase, is not functional and is unable to catalyze the NADPH-dependent reduction of glutamyl-tRNA. HemA deficient bacteria are unable to grow in the absence of 5-ALA and/or hemin. More particularly, "HemA deficient bacteria" are understood to be a bacteria that do not demonstrate significant growth (p <

0.05) of the cells in LB media within an 8 hour time period as determined by at optical density measurement at 600 nm (OD600). HemA deficient bacteria can be made from wild-type bacteria by techniques known in the art such as knocking-out, truncating, mutating or inactivating the native hemA gene such that it no longer expresses an active hemA protein. Inactivation can be achieved by mutation, deletion or inactivation of the coding and/or non-coding regions of the gene. Mutation can be achieved, for example, by site directed mutagenesis, or by any method known to those skilled in the art. Exemplary mutations of the HemA protein that cause a decrease or lack of detectable activity include, by way of non-limiting example, one or more of the following point mutations (numbered according to SEQ ID NO:2): G7D, C50S, R52Q,

G106N, E114K, S145F, G191D, R314C and combinations such as G44C/S105N/A326T and

S22L/S164F (Schauer et al., 2002). Knock-out of the hemA gene may be achieved for example by the methods described in Datsenko and Wanner, 2000 (incorporated herein by reference), as used to generate the bacteria strain E.coli BL21(DE3) AhemA, used in example 4. Inactivation can be achieved for example by the hemA41 mutation, which is present in the known hemA deficient, heme permeable, hemA E.coli mutant HU227 (available from CGCS Yale University, CGSC#8002) and the hemA E.coli mutant SASX41B (CGCS Yale University, CGSC#4806).

[54] The hemA deficient bacteria can be Gram positive or Gram negative. For instance, where a bacteria is Gram negative, it can be Escherichia coli {E.coli), for example strain K-12 (ATCC 10789), BL21(DE3) (ATCC BAA1025), DH5a (Invitrogen) or HK100(DE3) (Invitrogen, NB: DE3 signifies that the cells carry the lambda DE3 lysogen used to induce expression in TV- driven expression systems). Thus the hemA deficient bacteria strain can be one of the following three strains: E. coli BL21(DE3) AhemA, E. coli DH5a AhemA or E. coli HK100(DE3) AhemA.

[55] With regard to these three E.coli strains, BL21(DE3) AhemA is particularly suitable for recombinant protein expression, E. coli DH5a AhemA is particularly suitable for DNA preparation and classical cloning protocols, and E. coli HK100(DE3) AhemA is particularly suitable for ligase independent cloning procedures (PIPE cloning).

[56] The growth of untransformed hemA deficient bacteria requires that the growth medium contains a sufficient quantity of 5-ALA to support their growth. Figure 9 illustrates that at 37°C in LB medium at lOOOrpm, growth of the BL21(DE3) hemA mutant strain can occur when the concentration of 5-ALA is 2.5mg/L or greater. Similar experiments can be used to determine suitable concentrations for use with any bacteria of interest without undue burden.

Expression vector:

[57] The selection method of the invention comprises transforming hemA deficient bacteria with an expression vector suitable for complementing hemA deficient bacteria, comprising a functional hemA gene as a selection marker, wherein an active glutamyl tR A-reductase is expressed from said hemA gene. The terms "selection" and "selection marker" take the meaning known to those skilled in the art referring to the process of using a selection marker/selectable marker and a selection agent to identify host cells with specific genetic properties (e.g. that the host cell contains a vector such as a plasmid). More particularly, the term "selection marker" is understood to refer to a known coding sequence present in an expression construct, typically a vector, to complement a specific, known deficiency in a host cell to permit the host cell to grow under selection conditions that would otherwise not permit growth of the host cell and result in persistence of the vector in the cell. When grown under the appropriate selection conditions, the presence of the selection marker in the vector results in maintenance of the vector in the cell. The selection marker is preferably not an antibiotic resistance marker or an auxotrophic marker. In certain embodiments, the selection marker is expressed under the control of a weak promoter (e.g., the P3 promoter from the AmpC gene, Stauffer et al. 1986). In certain embodiments, the selection marker is expressed under the control of a constitutive promoter. In preferred embodiments, the selection marker is expressed under the control of a weak constitutive promoter. Other suitable promoters are known in the art, for example, from Papdakis et al. (2004), herein incorporated by reference.

[58] Any expression vector commonly used in the production of therapeutic products can be used, where the functional hemA gene is inserted into the vector using methods generally known in the art. Suitable vectors include episomal vectors, such as plasmids, for example, expression vectors derived from the pET24 expression vector (SEQ ID 7), as used in Example 1. The pHem expression vector (SEQ ID: 8) of Figure 1 is also suitable for the use in the selection method of the invention. The pHem vector of Figure 1 is 5416bp in length, but the expression vector can be of any size.

[59] The vector may be a circular or linear nucleic acid molecule capable of replication in a cell, and preferably is episomal, i.e. non-integrating. Other suitable vectors include, but are not limited to, phagemids, cosmids, and bacterial artificial chromosomes (BACs). Preferably the vector is a plasmid that replicates within a bacterial cell independently of the chromosomal DNA, i.e. extrachromosomally.

[60] The expression vector preferably comprises an origin of replication that is functional in the host bacterium. Any origin of replication commonly known to those skilled in the art can be used, for example Fl, or Ml 3, or ColEl origins of replication, as in Figure 1.

[61] The expression vector also will comprise a promoter or regulatory region for the expression of the functional hemA gene. The promoter or regulatory region can include a constitutive promoter or an inducible promoter. Many such bacterial promoters are commonly known to those skilled in the art, for example the LacO promoter. In one embodiment of the present invention, the hemA gene is under control of the weak constitutive P3 promoter from the ampC gene. A weak promoter is preferable in order to provide the appropriate quantity of the hemA protein for its metabolic role, without producing excess hemA. [62] The expression vector can also comprise a multiple cloning site (MCS) for insertion of a nucleic acid of interest. An MCS is a sequence having several restriction enzyme sites to facilitate insertion of a sequence of interest at this position in the vector. The multiple cloning site according to the present invention may, for example, be derived from the multiple cloning sites of any commercially available plasmid. In certain embodiments, the hemA gene is not inserted into the MCS.

[63] The expression vector can also comprise a gene for antibiotic resistance, such as the Kanamycin or ampicillin resistance genes, although this is not intended for use as a selection marker in the methods of the invention. For example the pET24 expression vector, and pET24 derived expression vectors, comprise a kanamycin resistance gene.

[64] The expression vector will usually include a nucleic acid of interest (but can also be provided without a nucleic acid of interest e.g. as a cloning vector in a kit ready for the introduction of a nucleic acid of interest e.g. for clone selection/library screening etc.). It is preferable that the nucleic acid of interest is under control of a different promoter to hemA, such that high levels of expression of the nucleic acid of interest can be achieved without impacting hemA levels. The nucleic acid of interest can be inserted into the expression vector at an MCS, such as the MCS shown in Figure 1.

Nucleic acids and proteins of interest:

[65] The nucleic acid of interest for example can be a gene of interest which encodes a protein that can be produced and isolated. As used herein, a "nucleic acid of interest" refers to a nucleic acid sequence, such as a DNA sequence, that encodes and is used to produce a product of interest typically for purification from the cell in which the product is expressed. In certain embodiments, the nucleic acid of interest encodes a polypeptide particularly a heterologous polypeptide, i.e., a protein that is not naturally expressed by the host bacterium by way of non- limiting example, bacterial proteins from other bacterial species, viral genes, eukaryotic genes, artificial polypeptide sequences, non-wild-type proteins, mutant proteins, truncated proteins and the like. In a preferred embodiment, the expression of the nucleic acid of interest is not required for the growth of the cell in which it is expressed. In certain embodiments, the nucleic acid of interest is expressed under the control of a strong promoter (e.g., a T7 promoter). In certain embodiments, the nucleic acid of interest is expressed under the control of an inducible promoter. In certain embodiments, the nucleic acid of interest is expressed under the control of a strong, inducible promoter. In certain embodiments, the nucleic acid of interest is expressed under the control of a weak, inducible promoter. The nucleic acid of interest can be for use in any bioprocess, for example, biochemical characterisation, biopharmaceutical research or protein crystallisation.

[66] In a preferred embodiment, the nucleic acid of interest does not encode an active glutamyl tR A reductase, i.e. it is separate from the hemA gene of the vector. More preferably, the nucleic acid of interest does not encode a protein involved in tetrapyrrole synthesis (including hemM). In certain embodiments, the nucleic acid of interest is a nucleic acid sequence from a prokaryotic organism. In certain embodiments, the nucleic acid of interest is a nucleic acid sequence from a prokaryotic organism other than E. coli. In certain embodiments, the nucleic acid of interest is a nucleic acid sequence from a eukaryotic organism. In certain embodiments, the nucleic acid of interest is an artificial sequence. In certain embodiments, the expression vector comprises a multiple cloning site. In certain embodiments, the nucleic acid of interest is inserted into the multiple cloning site in the vector. In certain embodiments the vector includes a promoter sequence to control the expression of the nucleic acid of interest. In a preferred embodiment, expression of the hemA gene and the nucleic acid of interest are under the control of separate promoters. In certain embodiments, at least one of the promoter sequence controlling the expression of the hemA gene and the promoter sequence controlling the expression of the nucleic acid of interest are from different organisms.

[67] In certain embodiments, the vector comprises a coding sequence for expression as a fusion construct with the nucleic acid of interest. In certain embodiments, the coding sequence for expression as a fusion construct with the nucleic acid of interest is a reporter construct. In certain embodiments, the coding sequence for expression as a fusion construct with the nucleic acid of interest is a tag to facilitate purification of the expression product of the nucleic acid of interest.

[68] In certain embodiments, the nucleic acid of interest can be for use in the preparation of a pharmaceutical composition, such as a vaccine. For example, the nucleic acid of interest can be a gene which expresses an immunogen. Example 6 demonstrates the high yielding expression of two model antigens, STA01 1 (disclosed in US8,632,783; GL88193872) and GBS1523 (Uniprot Accession No: Q8E479), by the selection method of the invention. Thus, particularly the nucleic acid of interest is a gene which codes for a protein of interest such as an immunogen or antigen. An antigen refers to a molecule containing one or more epitopes (e.g., linear, conformational or both) capable of eliciting an immunological response, more particularly a protective immune response, when administered to a subject such as an animal. By "elicit" is meant to induce, promote, enhance or modulate an immune response or immune reaction. A preferred protein is CRM 197 (NCBI protein Accession number 1007216A), for example, expressed from a nucleic acid sequence of interest such as nucleic acid sequence NCBI Accession number HW071379 and described in EP2445930.

[69] Alternatively, the nucleic acid of interest can express a reporter protein such as blue fluorescent protein (BFP) or green fluorescent protein (GFP), as in example 4. The nucleic acid of interest can be inserted into the expression vector at a MCS, such as the MCS shown in the pHem vector of Figure 1. For example, a reporter gene such as BFP or GFP (SEQ ID NO: 10) can be inserted at the MCS of pHem to provide pHem-BFP (SEQ ID NO: 9) or pHem-GFP (SEQ ID NO: 13) respectively. In certain embodiments, the reporter protein can be an enzymatic reporter construct including, but not limited to, horse radish peroxidase (HRP), luciferase, or alkaline phosphatase.

[70] In certain embodiments, the expression vector can include a coding sequence for an affinity tag or an epitope tag adjacent to the MCS to allow for generation of a fusion protein with the expression product of the nucleic acid of interest to facilitate detection or purification of the polypeptide expressed from the nucleic acid of interest. Such tags include, but are not limited to a 6 X His tag, a GST-tag, an HA1 tag, and a myc tag.

[71] The protein of interest is not expressed as a fusion to the glutamyl tRNA reductase - it is an entirely separate molecule. Therefore the protein of interest, whilst it may be a reporter or contain sequences that assist in purification, these are not produced as part of the glutamyl tRNA reductase.

[72] In particular embodiments, the nucleic acid of interest can be isolated and purified, for example for use in the preparation of a pharmaceutical composition, such as a vaccine. The nucleic acid of interest can take the form of DNA or RNA, but usually it will be DNA. For instance, in one embodiment the nucleic acid of interest is an expression vector of the invention and the invention therefore provides a method of producing and purifying the expression vector which is propagated by the selection method of the invention. Media:

[73] Any growth medium suitable for culturing bacteria, but which does not support growth of hemA deficient bacteria may be used for the selection and culturing of transformed bacteria. HemA deficient bacteria are unable to grow in the absence of 5-ALA and/or hemin. In one embodiment the medium lacks sufficient 5-aminolevulinc acid (5-ALA) and/or hemin to support growth of the hemA deficient bacteria. For example, complex media such as LB broth (G.Bertani, 1951), as demonstrated in the examples, or Terrific Broth (TB) (Tartoff and Hobbs. 1987) are suitable.

[74] Figure 9 illustrates the effect of varying 5-ALA concentration on the growth rate of hemA deficient BL21(DE3) E.coli. The growth rate was shown to decrease with lower 5-ALA concentrations. The Figure shows that at 37°C in LB medium at lOOOrpm, growth is restricted when the concentration of 5-ALA is lower than 2.5mg/L. This indicates that complex growth media such as LB broth, with a 5-ALA concentration lower than 2.5 mg/L, will be suitable for the selection and culturing of this strain after transformation. Similar experiments will reveal a suitable concentration for use with any bacteria of interest. As used herein, the term "lacks sufficient 5-aminolevulinc acid (5-ALA) to support the growth of the hemA deficient bacteria" refers to a level of 5-ALA that does not exceed 2.5mg/L, and particularly is below 2.5mg/L, more particularly below 2mg/L, yet more particularly below 1.5mg/L, still yet more particularly below l .Omg/L, below 0.5mg/L, below O. lmg/L, below 0.05mg/L, below O.Olmg/L or essentially free of 5-ALA, i.e. trace or undetectable amounts about O.Omg/L or any range there between. The term "lacks sufficient hemin to support the growth of a hemA deficient bacteria" refers to a level of hemin that does not exceed 4mg/L and particularly is below 4mg/L, more particularly below 3mg/L, yet more particularly below 2mg/L, still yet more particularly below l .Omg/L, below 0.5mg/L, below O. lmg/L, below 0.05mg/L, below O.Olmg/L or essentially free of hemin, i.e. trace or undetectable amounts about O.Omg/L or any range there between.

[75] By "complex media" is meant media wherein the exact composition and concentration of their components is not known. Complex media are typically derived from, for example, partially digested yeast, beef, soy, and/or additional proteins, and due to their rich array of nutrients are suitable for efficient growth of bacteria. Chemically defined media are also suitable for the use of the invention. By "defined media" or "chemically defined media" is meant media wherein the exact composition and concentration of substantially all of the components of the media is known. Transformation:

[76] The method for selection of transformed bacteria involves transforming heniA deficient bacteria, with a vector carrying a functional hemA gene, as a selection marker. Transformation refers to the insertion of an exogenous polynucleotide into a host cell, which can be for example a competent cell, irrespective of the method used for insertion, for example, transformation by direct uptake, transfection, infection, and other methods known in the art are suitable. By way of non-limiting example, such methods for direct DNA introduction may include calcium phosphate co-precipitation, electroporation, lipofection, and microinjection. The exogenous polynucleotide may be maintained as a non-integrated vector, for example, a plasmid or an episome, or alternatively, may be integrated into the host genome. As used herein the term "transformed bacteria" denotes host bacteria that have been genetically engineered to produce one or more heterologous polypeptides under the control of one or more promoter-operators. As disclosed herein, in particular embodiments, a first heterologous polypeptide is the hemA protein (glutamyl tR A-reductase) and a second heterologous polypeptide is a protein of interest. Such bacteria are sometimes referred to herein as "transformants".

Downstream uses of expressed proteins:

[77] The method of selection is suitable for the propagation of the nucleic acid of interest. Propagation of the nucleic acid of interest is achieved by replication of the expression vector, which results in an increase in the copy number of the nucleic acid of interest in the transformed bacteria. The selection method of the invention provides a high plasmid copy number, as demonstrated in example 2. In addition, the method of selection is especially useful for the recombinant protein production. Examples 4 and 6 demonstrate the high yield of recombinant protein expression provided by the invention, and the stability of expression over many cultivation cycles. In particular, example 6 shows the high yield of antigen expression that can be achieved by the methods of the invention. Particularly plasmid copy number in AhemA transformants is more than 40, more than 50, more than 60, more than 70, more than 75, more than 80, more than 85, more than 90, more than 95, more than 100, more than 110, more than 120, more than 130, more than 140, more than 150, more than 160, more than 170, more than 180, more than or about 190, about 200, or any range between these Figures. Particularly plasmid copy number in AhemA transformants is stable for at least 5 passages, more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 passages, about 20 passages, in the absence of antibiotic. In particular embodiments, plasmid DNA yield in AhemA transformants is at least about 1.5, 2, 3 or 4 times higher than hemA transformants grown in the presence of antibiotic.

[78] The transformed bacteria of the invention can be used for production of the nucleic acid of interest or for production of a protein of interest expressed by the nucleic acid of interest, i.e. a nucleic acid sequence encoding a protein of interest. The protein or nucleic acid can then be isolated. The invention also provides a method for preparing a pharmaceutical composition, comprising isolating the nucleic acid of interest, or the protein expressed by the nucleic acid of interest, and preparing a pharmaceutical composition comprising the isolated nucleic acid or protein.

[79] The term "pharmaceutical composition" according to this invention refers to the nucleic acid of interest or protein isolated and purified according to this invention, and suitable for in vivo administration, for example for use as a vaccine in immunising subjects against various diseases. These compositions will typically include the protein expressed by the nucleic acid of interest and any number of pharmaceutically acceptable excipients.

Kits:

[80] The present invention provides kits for use in the selection method. Kits comprise hemA deficient bacteria and an expression vector of the invention. Optionally, kits may also contain media for the growth of hemA deficient bacteria and/or media for the growth of transformed bacteria, as well as instructions for performing the methods of the invention.

General:

[81] The term "comprising" encompasses "including" as well as "consisting" e.g. a composition "comprising" X may consist exclusively of X or may include something additional e.g. X + Y. The term "consisting essentially of means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do no materially alter the basic and novel characteristics of the claimed composition, method or structure. The term "consisting of is generally taken to mean that the invention as claimed is limited to those elements specifically recited in the claim (and may include their equivalents, insofar as the doctrine of equivalents is applicable). [82] The term "about" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[83] The word "substantially" does not exclude "completely" e.g. a composition which is "substantially free" from Y may be completely free from Y. For example, "substantially free" from Y can be understood as a composition containing not more than 5% Y, not more than 4% Y, not more than 3% Y, not more than 2% Y, not more than 1% Y, or not more than 0.1% Y. Where necessary, the word "substantially" may be omitted from the definition of the invention.

[84] As used herein, unless otherwise clear from context the term "or" is understood to be inclusive and can be used interchangeably with the term "and/or".

[85] A "host cell" is a cell which has been transformed, or is capable of transformation, by an exogenous DNA sequence. A host cell can be used, for example, for expression of a nucleic acid of interest or propagation of a plasmid vector or both.

[86] All GenBank Accession numbers provided herein are incorporated by reference in the version available on the date of filing the instant application.

MODES OF CARRYING OUT THE INVENTION

Example 1 - 5-ALA auxotrophy validation

[87] In order to validate the hemA selection method, a pET24 derived expression vector, pHem-BFP, was constructed. The vector expresses the wild type copy of the hemA gene, under the control of a weak promoter (P3 promoter from ampC gene) and the reporter gene blue fluorescent protein (BFP).

[88] In order to confirm the complementation system, the pHem-BFP vector was transformed into the K12 derived AhemA HU227 E.coli strain purchased from CGSC (Yale University).

[89] Figure 2 shows that the AhemA mutant is not able to duplicate in the absence of 5-ALA

(5-amino-4-oxo-pentanoic acid, CAS Number: 106-60-5) supplementation in both defined media

(M9+glucose) and complex media (LB) and that the pHem-BFP vector can effectively complement the auxotrophy and restore growth. If necessary, AhemA mutants can be propagated in the absence of vectors expressing a wild type hemA gene by supplementation with 5-ALA, i.e. for "empty cell" propagation.

[90] In comparison to the developed selection method, other auxotrophy complementation systems were evaluated. In particular the 4 amino acid auxotrophic K12 derived E. coli mutant strains purchased from CGSC (Yale University) shown in table 1.

Table 1

[91] These amino acid auxotrophic strains were transformed with complementation plasmids. The result of the complementation of these amino acid auxotrophic strains is shown in Figure 3. The mutant strains were grown and growth monitored in defined and complex media. The relevant vector, e.g. pArgH-GFP was added to some samples and to other samples the auxotrophic amino acid was added. The growth of these samples was also monitored in defined and complex media. It can be seen from Figures 3 a and 3b that the complementation selection used is able to effectively restore the growth of mutant strain in defined medium, but all tested amino acid auxotrophic strains are able to duplicate in complex medium in the absence of complementation. This makes these amino acid auxotrophy complementation selection methods ineffective when conducted in complex media, as the media provides enough of the required auxotrophic nutrients. In these experiments, cultivation was carried out at 37°C in 1 mL of M9 medium + glucose 0,4% w/v (defined) or LB (complex) in 96 Deep Well plates. Example 2 - Plasmid copy number maintenance

[92] The plasmid copy number (PCN) in the 5 -ALA complementation system (E.coli AhemA HU227 + pHem-GFP) was compared to that of the conventional antibiotic system (E.coli BL21 (DE3) + pET24 in the presence of kanamycin 50 mg/1 in complex medium LB). Plasmid copy number was calculated using quantitative PCR, as described in (Skulj et al, 2008). Table 2 shows that a high plasmid copy number is maintained compared to the conventional antibiotic based system. The sequence of pET24-GFP is provided as SEQ ID NO: 14.

[93] In a separate experiment, plasmid copy number for pHem/BL21(DE3) AhemA was measured over 20 passages and compared to the plasmid copy number exhibited by pET24- GFP/BL21(DE3) and pET24-GFP/BL21(DE3) with kanamycin. Plasmid copy number was calculated using quantitative PCR, as described in Skulj et al., 2008. Table 3 demonstrates that a high plasmid copy number is achieved in the hemA mutant strain compared to the wild type grown in the presence of antibiotic. Table 3 also shows that plasmid copy number for pHem/BL21(DE3) AhemA is stable after 20 passages without further selection. Each passage corresponds to a 1 : 100 dilution of the culture in fresh LB medium.

Table 3

[94] Plasmid copy number was also determined for two further hemA deficient E. coli strains, DH5a AhemA and E. coli HK100(DE3). After 1, 5, 10, 15 and 20 passages the PCN for these two strains was determined by quantitative PCR. Table 4 shows that these two hemA mutant strains also exhibit high PCN after 20 passages without further selection.

Table 4

Example 3 - Plasmid DNA production

[95] Plasmid DNA (pDNA) production was tested with respect to the two hemA deficient E.coli strains DH5aAhemA and HK100(DE3)AhemA. The hemA deficient and wild type strains were precultured overnight in either 2ml LB or 15ml LB. When an optical density at 600nm of 0.05 was reached, the strains were cultured for 16h at 37°C. Plasmid DNA was then extracted with OmegaBiotek Miniprep kit according to the manufacturer's instructions.

[96] Table 5 shows plasmid DNA yield in the DH5a hemA mutant strain is about 1.5-fold higher compared to the wild type grown in the presence of antibiotic, and two-fold higher in comparison to the wild type grown without selection. Similarly, table 6 shows plasmid DNA yield in the HK100(DE3) hemA mutant strain is about two-fold higher compared to the wild type grown in the presence of antibiotic and four-fold compared to the wild type grown without selection. Table 5 - micrograms of pDNA pHem/DH5 aAhemA pET24/DH5a pET24/DH5 + kanamycin

2ml 1.3 0.62 1.07

15ml 9.4 4.6 6.37 Table 6 - micrograms of pDNA

Example 4 - Recombinant protein production

[97] Analysis of recombinant protein production (RPP) in 5 -ALA complementation system required the generation of an E. coli (DE3) mutant strain in which the hemA gene was knocked out. E. coli BL21(DE3) hemA::kan mutant strain was constructed according to Datsenko and Wanner (Datsenko and Wanner, 2000, PNAS 97(12): 6640-5, incorporated herein by reference). The hemA deficient mutant and the corresponding BL21(DE3) wild type were transformed with the expression vector pHem-BFP and both recombinant strains were cultivated in standard conditions. Cultures were performed at 37 °C, 200 rpm in 100 ml shake flask with 20ml LB medium (E. coli BL21(DE3)/pHem-BFP in the presence of 50 mg/L kanamycin). Induction was performed at OD600nm 0,5-0,6 with ImM IPTG. Cells were harvested 4h post-induction. The whole cell pellet was washed 2x in PBS and resuspended in lOOmL PBS and confirmed by SDS- PAGE.

[98] The RPP was then evaluated by quantification of BFP fluorescence emitted by whole cells and confirmed by SDS-PAGE. The results can be seen in Figure 4, which shows that the 5- ALA complementation system produces a higher amount of recombinant protein compared to the standard antibiotic based selection method.

[99] In order to test the stability of recombinant protein production by the 5 -ALA method,

E. coli BL21(DE3) hemA::kan/pHem-BFP and E. coli BL21(DE3)/pHem-BFP were cultivated without antibiotic selection for a number of generations. Each generation corresponds to a 1 : 100 dilution of the culture in fresh LB medium. After 1, 2, 5, 10 and 15 generations the recombinant protein expression was analysed. The results in Figure 5 shows that BFP production in the hemA deficient strain is stable after 15 cultivation cycles without antibiotic while in the wild type strain it dramatically decreases after 5 cultivation cycles without selection. [100] Recombinant protein expression over many generations was also measured for GFP. E. coli BL21(DE3) AhemA/pHem-GFP and E. coli BL21(DE3)/pET24-GFP both with and without antibiotic selection, were cultivated for a number of passages. Each passage corresponds to a 1 : 100 dilution of the culture in fresh LB medium (1 passage ~ 7-8 duplications). After 1, 5, 10, 15 and 20 passages the recombinant protein expression was analyzed by measuring the specific fluorescence of GFP. Figure 8 shows that GFP production in the hemA mutant strain is stable after 20 cultivation cycles without antibiotic. In contrast, GFP production in the wild type strain dramatically decreases after 5 cultivation cycles without selection.

Example 5 - Mutant/wild type growth profiles

[101] The growth of the BL21(DE3) AHemA mutant strain was compared to that of the wild type. Figure 7a shows the comparison of growth profiles of BL21(DE3) for (i) the Ahem mutant complemented with pHem (ii) the wild type with pET24 and (iii) the wild type with pET24 and where kanamycin is added. Growth conducted at 37°C and 600rpm in 96 flat bottom wells in LB broth. Growth was measured according to optical density (LogOD600nm). The results in Figure 7a show that the AhemA mutant strain specific growth rate is comparable to the wild type. Growth of the AhemA mutant strain showed a shorter lag phase and higher biomass yield compared to the wild type in both the presence an absence of antibiotics.

[102] In addition, the growth of two further hemA deficient strains was monitored using the same method. Figure 7b shows the growth profile of pHem/DH5 AhemA in comparison to the wild type and Figure 7c shows the growth profile of pHem/HK100(DE3)AhemA in comparison to the wild type. Both of these hemA deficient strains show a shorter lag phase compared to the wild type and similar biomass yields.

Example 6 - Antigen expression

[103] The yield of antigens expressed by hemA deficient bacteria complemented with pHem was compared to the yield obtained for antigen expression in pET24/BL21(DE3). The yield was assessed for two model antigens, ST AO 11 (SEQ ID NO: 11), which is expressed strongly, and GBS1523-80 (SEQ ID NO: 12), a protein which is expressed with low yield in conventional E. coli expression. Both proteins were N-His tagged. Standard HT platform expression conditions (Enpresso system, Biosilta) were used, and there was no antibiotic addition. Immobilized metal affinity chromatography (IMAC) was utilised for purification. [104] The results presented in table 9 show that a twofold greater protein yield for STA011 was achieved by the pHem/AhemA system compared to standard conditions, and that a fourfold greater protein yield for GBS 1523-80 was achieved in the pHem/AhemA system compared to standard conditions. The sequences of pHem-STAOl l and pHem-GBS 1523-80 are provided as SEQ ID NOs: 15 and 16 respectively.

Table 9 - Protein yield (mg/g)

Example 7 - CRM197 expression

[105] The yield of antigens expressed by hemA deficient bacteria complemented with pHem is compared to that obtained for antigen expression in pET24/BL21(DE3). The yield is assessed for CRM 197 (the enzymatically inactive and nontoxic form of diphtheria toxin that contains a single amino acid substitution atG52E) by hemA deficient bacteria complemented with pHem. The protein is a HisTag-CRM197 fusion protein expressed from pHem-CRM197 (SEQ ID NO: 17). Standard HT platform expression conditions (Enpresso system, Biosilta) can be used without antibiotic addition. Immobilized metal affinity chromatography (IMAC) can be utilised for purification.

[106] Whilst the invention has been exemplified using E. coli, it will be apparent to those skilled in the art that the invention may be applied more broadly to other bacteria such as yeast and other commonly used bacteria.

[107] It will be understood that all features referred to in one embodiment or aspect described above can be applied mutatis mutandis to other embodiments and aspects. REFERENCES

[1] Levy, S.B., J. Antimicrob. Chemother. 49 (2002) 25-32

[2] Chen et al (1994), J Bacteriol 176(9): 2743-2746.

[3] Datsenko and Wanner, 2000, PNAS 97(12): 6640-5

[4] G.Bertani, J. Bacteriol. 1951, 62(3):293.

[5] Tartoff and Hobbs. 1987. Bethesda Research Laboratories Focus 9: 12.

[6] Skulj et al, Microbial Cell Factories 2008, 7-6

[7] Stauffer LT, Plamann MD, Stauffer GV, Gene 1986, 44:219-226.

[8] Papadakis ED, Nicklin SA, Baker AH, White SJ, Current Gene Therapy, 2004, Vol. 4, No. 1.

[9] Schauer et al. J Biol Chem. 2002 Dec 13;277(50):48657-63.

Claims

1. A method for antibiotic-free selection of transformed bacteria comprising the steps of:

i) transforming a bacterial cell lacking a functional hemA gene with a non-integrative vector comprising a first nucleic acid sequence encoding a functional glutamyl tRNA- reductase and a second nucleic acid sequence encoding a protein of interest to give a transformed bacterial cell and;

ii) growing the transformed bacterial cell in a growth medium that does not support growth of bacterial cells lacking a functional hemA gene in the absence of the vector.

2. The method of claim 1 wherein the growth medium comprises less than 2.5mg/L of 5- aminolevulinc acid (5 -ALA) and/or less than 4mg/L of hemin.

3. The method of claim 1 or claim 2, wherein the growth medium is a complex medium.

4. The method of any one of the preceding claims, wherein the bacterial cell lacking a functional hemA gene contains at least one mutation in its hemA gene selected from the group consisting of G7D, C50S, R52Q, G106N, E114K, S145F, G191D, R314C, G44C/S105N/A326T and S22L/S164F.

5. The method of any one of claims 1 to 3 wherein,

(i) the bacterial cell is selected from the group consisting of Escherichia coli (E.coli) strains SASX41B, HU227 and EV61; or

(ii) wherein the hemA gene of the bacterial cell lacking a functional hemA gene has been knocked out or is a partial knock-out; and/or

(iii) wherein the bacterial cell lacking a functional hemA gene is a strain of E.coli selected from the group consisting of BL21(DE3) AhemA or DH5alpha AhemA and HK100(DE3) AhemA.

6. A non-integrative expression vector comprising (i) as a selectable marker, a first nucleic acid sequence encoding a functional glutamyl tRNA-reductase, operably linked to a first regulatory control sequence that directs expression of the glutamyl tRNA-reductase in a bacterial host cell; (ii) a multiple cloning site (MCS) and (iii) a second regulatory control sequence operably linked to the MCS such that a second nucleic acid of interest inserted into the MCS will be expressed, separately from the glutamyl tRNA-reductase, in a bacterial host cell comprising the expression vector,

wherein the expression vector does not encode a polypeptide involved in tetrapyrrole synthesis other than glutamyl tRNA-reductase.

7. The expression vector of claim 6, wherein the vector is derived from a pET24 plasmid.

8. The expression vector of claim 6 or claim 7, further comprising a second nucleic acid sequence encoding a protein of interest inserted into the MCS, wherein the second nucleic acid of interest does not encode a polypeptide involved in tetrapyrrole synthesis.

9. The expression vector of claim 8 wherein the protein of interest is a therapeutic polypeptide and/or immunogenic.

10. A bacterial cell lacking a functional hemA gene which cell comprises the expression vector of claims 6 or claim 7, wherein the functional glutamyl tRNA-reductase of the expression vector complements hemA deficiency in the cell.

11. A bacterial cell lacking a functional hemA gene which cell comprises the expression vector of claim 8 or claim 9, wherein the functional glutamyl tRNA-reductase of the expression vector complements hemA deficiency in the cell.

12. A kit containing (i) a bacterial cell lacking a functional hemA gene and (ii) the expression vector of any of claims 6 to 9.

13. Use of the vector of claim 6 or claim 7 in an antibiotic-free method of selection of transformed bacteria.

14. Use of the vector of claim 8 or claim 9 in an antibiotic-free method of producing a protein of interest.

15. A method of producing a protein of interest, comprising a step of growing the bacterium of claim 11 under conditions such that (a) the growth of a bacterial cell lacking a functional hemA gene is not supported in the absence of a complementary hemA gene, and (b) the protein of interest is produced.