WO2008025744A1 - Extra-cellular production of recombinant proteins - Google Patents

Abstract

The invention is related to a method for extracellular production of proteins in alanine racemase deficient bacteria strains wherein leakage of the expressed target protein to the culture medium through the cell wall membrane is triggered by repression of the cystathionine beta- lyase (MetC) encoding gene.

Description

EXTRA-CELLULAR PRODUCTION OF RECOMBINANT PROTEINS

FIELD OF THE INVENTION

The present invention relates to a method for controlled secretion of recombinant proteins using a genetically modified bacteria strains.

BACKGROUND OF THE INVENTION

Secretion of recombinant proteins to the culture medium of bacteria, in particular E. coli has several advantages over intracellular production. These advantages include less contamination of product by host proteins, reduced proteolytic activity, enhanced protein fold- ing capacity, and higher product stability and solubility. E. coli normally does not secrete proteins to the extracellular environment except for a few classes of proteins such as toxins and hemolysin. Therefore efficient pathways for translocation through the outer membrane are absent in E. coli. In most cases, protein secretion to the medium occurs by leakage of perip- lasmic contents. Passive transport across the outer membrane can be stimulated by external or internal destabilization of the E. coli structural components. Destabilization is achieved either by lysis proteins working from the interior of the cell, by using strains lacking structural membrane components or by permeabilization directed from the cell exterior either mechanically, enzymatic or chemically ( WO2001066776A3).

Although the current strategies employed to increase the permeability of the outer membrane of bacteria used for heterologous expression of target proteins proved to be success in enhancing extra-cellular production of recombinant proteins in E. coli at laboratory scale, they are usually not suitable for industrial production. Biochemical and physical methods can be applied only after harvesting cells, and are not easily integrated with cultivation. The strains with destabilized outer membrane normally are growth impaired and lack the necessary robustness for high-density fermentations. An expression/secretion system in which secretion can be regulated during growth is thus highly desirable and provided by the present invention.

SUMMARY OF THE INVENTION

In its broadest aspect the present invention is related to a method for extracellular production of proteins in alanine racemase deficient bacteria strains wherein leakage of the expressed target protein to the culture medium through the cell wall membrane is triggered by repression of the beta-cystathione lyase (MetC) encoding gene. An alanine racemase deficient strain is a strain which cannot grow without addition of D-alanine to the growth medium.

Repression of the cystathionine beta-lyase (MetC) encoding gene means that the alanine racemase activity of the gene product is sufficiently reduced to trigger leakage of the expressed product to the culture medium.

With MetC is meant the enzyme previously characterized as a cystathionine beta- lysase (Belfaiza J., Parsot C, Martel A., de Ia Tour CB, Margarita D., Cohen GN, Saint- Girons I:" Evolution in biosynthetic pathways: two enzymes catalysing consecutive steps in methionine biosynthesis originate from a common ancestor and posses a similar regulatory region; Proc. Natl. Acad. Sci. U.S.A 83: 867-871 (1986)

In one embodiment of the invention the cystathionine beta-lyase (MetC) derived alanine racemase activity is 100% reduced.

However, sufficient leakage may be obtained even if part of the alanine racemase activity is maintained. Thus, in another embodiment of the invention 5-10%, 10-20%, 20- 30%, 30-40% or even up to 50% of the alanine racemase activity may be maintained.

Repression of cystathionine beta-lyase (MetC) will happen at high L-methionine content. Thus in one embodiment the invention is related to a method for making proteins in alanine racemase deficient bacteria strains wherein the secretion of the target protein from the cell to the culture medium is triggered by addition of L-methionine to the culture medium at an appropriate time during the cultivation and in one embodiment of the invention L- methionine or a functional analogue thereof is added to the culture medium after an initial growth phase and in particular when a high cell density has been obtained.

When L-methionine or an analogue thereof with the same effect is added to the culture medium the MetC gene will be negatively regulated by the methionine repressor MetJ. MetJ is a transcriptional repressor controlling several genes involved in methionine biosynthesis (Fig. 1 ). It represses the metA, metC, metE and metF and other genes from the Methionine pathway through dimerization, interaction with the corepessor S-adenosyl methionine (SAM) and binding to regulatory DNA (Met box regions) (Saint-Girons I., Duchange N., Cohen GC, Zakin, MM; (1984) J. Biol., Chem 259: 14282-14285). When MetC is repressed it will not exert its newly discovered D-alanine producing capacity and we have found that the alanine racemase deficient cells then will start leaking the expressed target protein to the culture medium.

To get more robust alanine racemase deficient production cells in the initial growth phase it may be advantageous that the MetC expression is boosted in this phase causing production of D-alanine to give a more robust cell wall. Thus in one embodiment of the invention the host cell is transformed with a suitable plasmid expressing MetC under regulation of its own operator region

In this case repression of the MetC gene will require greater amounts L-methionine to trigger leakage of the target protein compared to a cell which contains no extra copies of the MetC gene.

It will be within the skills of the expert in the art to determine the L-methionine amount necessary for triggering leakage of the target product to the culture medium.

A variant of this embodiment is to integrate one or more copies of the cystathionine beta-lyase (MetC) expression cassette under regulation of its own operator region into the chromosome of the host cell. The integration of the MetC cassette on the chromosome should not have a negative influence on the natural genes on the chromosome. Thus in one embodiment of the invention the MetC cassette is integrated in the original positions of the air or alr/dadx gene.

A further variant of this embodiment is to integrate the air gene or the dadx gene under the control of an operator region capable of reacting with the repressor MetJ into the host cell genome.

Examples of suitable operator regions are the MetC operator region, the MetE operator region and the MetF operator region. Such operator regions may be optimized to have reduced affinity for MetJ binding (Wild, J. ,McNaIIy. T., Phillips, S. E. V., Stockley, P. G., Mo- lecular Microbiology (1996) 21 (6); 1 125-1135).

According to a further embodiment of the invention the host cell may carry a mutation in the MetJ gene rendering MetJ partially functional.

A partly functional gene in this context is a MetJ which has retained at least 10% of its original biological activity. In another embodiment of the present invention the MetJ gene will have retained at least 10-20% of its original biological activity.

In further embodiment of the present invention the MetJ gene will have retained at least 20-30% of its original biological activity.

In further embodiment of the present invention the MetJ gene will have retained at least 30-40% of its original biological activity.

In further embodiment of the present invention the MetJ gene will have retained at least 40-50% of its original biological activity.

In further embodiment of the present invention the MetJ gene will have retained at least 50-60% of its original biological activity. In further embodiment of the present invention the MetJ gene will have retained at least 60-70% of its original biological activity.

In further embodiment of the present invention the MetJ gene will have retained at least 70-80% of its original biological activity. In further embodiment of the present invention the MetJ gene will have retained at least 80-90% of its original biological activity.

The added amount of L-methionine and the suitable time intervals for addition will vary depending on which of the above embodiments of the invention are used. However it will be within the knowledge of the expert within the art to determine suitable amounts and time intervals for addition by simple testing when leakage of the target protein is triggered.

Thus the L-methionine or an analogue thereof may be added at intervals in amounts of from about 0.1 to about 10 mM, from about 0.1 to about 8 mM, from about 0.1 to about 6 mM, from about 0.1 to about 4 mM or from about 0.1 to about 2 mM.

The interval at which the L-methionine is added may also vary. In one embodiment of the invention L-methionine or an analogue thereof is added in at intervals of 1-20 hours, 1-18 hours, 1-16, 1-14, 1-12, 1-10, 1-8, 1-6, 1-4 or 1-2 hours.

In one embodiment of the invention L-methionine or an analogue thereof is added in amounts of from about 0.1 to about 10 mM at intervals of 1-20 hours.

In another embodiment of the invention L-methionine or an analogue thereof is added in amounts of from about 0.1 to about 8 mM at intervals of 1-20 hours.

In another embodiment of the invention L-methionine or an analogue thereof is added in amounts of from about 0.1 to about 6 mM at intervals of 1-20 hours.

In another embodiment of the invention L-methionine or an analogue thereof is added in amounts of from about 0.1 to about 4 mM at intervals of 1-20 hours. In another embodiment of the invention L-methionine or an analogue thereof is added in amounts of from about 0.1 to about 2 mM at intervals of 1-20 hours.

In one embodiment of the invention L-methionine or an analogue thereof is added in amounts of from about 0.1 to about 10 mM at intervals of 1-10 hours.

In another embodiment of the invention L-methionine or an analogue thereof is added in amounts of from about 0.1 to about 8 mM at intervals of 1-10 hours.

In another embodiment of the invention L-methionine or an analogue thereof is added in amounts of from about 0.1 to about 6 mM at intervals of 1-10 hours.

In another embodiment of the invention L-methionine or an analogue thereof is added in amounts of from about 0.1 to about 4 mM at intervals of 1-10 hours. In another embodiment of the invention L-methionine or an analogue thereof is added in amounts of from about 0.1 to about 2 mM at intervals of 1-10 hours.

In a further embodiment L-methionine or an analogue thereof is added in amounts of from about 0.1 to about 2 mM at intervals of 2-5 hours. In a further embodiment L-methionine or an analogue thereof is added in amounts of from about 1.0 to about 2 mM at intervals of 2-5 hours.

In a further embodiment L-methionine or an analogue thereof is added in amounts of from about 1.25 to about 2 mM at intervals of 2-5 hours.

In a further embodiment L-methionine or an analogue thereof is added in amounts of from about 1.5 to about 2 mM at intervals of 2-5 hours.

Repression of the MetC encoding gene may also be effected by manipulating the MetJ regulon/operon region so that the MetJ repressor is no longer influenced by the content of L-methionine or an analogue thereof with the same effect.

Thus in one embodiment of the invention the MetJ is under regulation of the same promoter/operator which is used for expression of the target protein enabling the cell to repress MetC and induce leakage from the periplasm simultaneously with induction of expression of the recombinant protein destined for secretion.

By periplasm is meant the substance which is placed in the space between the plasma membrane and the outer membrane (periplasmic space) in gram-negative bacteria. Induction of protein expression in bacteria is well known. In the present invention the induction of protein expression is advantageously made by addition of IPTG (isopropyl-β-D- thiogalactopyranoside).

A well suited promoter is the tightly regulated T7/lacl promoter. Thus in another embodiment of the invention the methionine repressor cassette in the alanine racemase defi- cient strain is replaced with an expression cassette in which the wild type MetJ gene is under the control of the T7/lacl promoter.

The present invention may make use of any bacteria strain which can be made alanine racemase deficient. However the present invention will typically make use of E. coli as host strain. Thus in one embodiment of the method according to the invention the host strain is

E. coli.

As explained and well known within the art alanine racemase deficiency in bacteria may be introduced by deletion mutations in a single alanine racemase gene. In other bacteria deletion mutations of more than one alanine reacemase gene is necessary to obtain an fully D-alanine auxotrophic strain and in one embodiment of the invention the host cell has a double knockout of both alanine racemase genes.

In a further embodiment of the invention the double knock out is an E. coli dadX/alr double knock out. In another embodiment of the invention the protein of interest is targeted to the E. coli periplasm by means of an N-terminal signal sequence.

The targeting of the protein of interest to the periplasm will ease the subsequent leakage to the culture medium.

DESCRIPTION OF THE DRAWINGS Figure 1 illustrates the methionine pathway showing the relations between the Met repressor (MetJ) and MeU responsive genes and the link to D-alanine and peptidoglycan synthesis via MetC. Stippled arrows indicate activator action and abrupted stippled lines indicate repressor action;

Figure 2 shows a growth curve displaying growth characteristics of BL21 (DE3)dadX /air (in OD600) during a overnight cultivation with different amounts of L-methionine added to the defined medium;

Figure 3 shows a schematic description of the alkaline phosphatase expression plasmid (phoAm_pET39b);

Figure 4 shows the level of alkaline phosphatase in growth medium and in periplas- mic fraction respectively at different L-methionine feedings. In figure 4 the symbols have the following meaning:

A1 : Adding L-Met at 0.01 mM

A2: Adding L-Met at 0.01 mM, feeding twice every 3hrs

A3: Adding L-Met at 0.01 mM, feeding twice every 4hrs B1 : Adding L-Met at 0.05mM

B2: Adding L-Met at 0.05mM, feeding twice every 3hrs

B3: Adding L-Met at 0.05mM, feeding twice every 4hrs

C1 : Adding L-Met at 0.1 mM

C2: Adding L-Met at 0.1 mM, feeding twice every 3hrs C3: Adding L-Met at 0.1 mM, feeding twice every 4hrs

D1 : Adding L-Met at 0.2mM

D2: Adding L-Met at 0.2mM, feeding twice every 3hrs

D3: Adding L-Met at 0.2mM, feeding twice every 4hrs

E phoA-pET39b/ BL21 (DE3)dadXVa/r without L-Met F phoA-pET39b/BL21 (DE3) without L-Met

GM: growth medium Peri: periplasm

Figure 5 shows growth curves of strain BL21 (DE3)alr-::MetCOPdadx- and BL21 (DE3)alr-dadx- with or without inhibitor L-Met during eight hours cultivation. Kl stands for strain BL21 (DE3)alr-::MetCOPdadx- and DKO stands for strain BL21 (DE3)alr-dadx-.The inhibitor methoinine was added at concentration of OmM, 0.1 mM, 1 mM and 5mM respectively; and

Figure 6 shows the western blot results using anti-mouse Fab antibody. Samples were medium supernatant from different fermentation tanks with different strains during different induction period. They were separated by 8-15% gel at non-reduced conditions. The upper band with the molecular weight of 150KDa is the target protein Ly49-5E6 F(ab')2-LZ. Other two bands with molecular weight of around 50KDa and 25KDa are Fab fragment and Light chain respectively. In figure 6 the symbols have the following meaning: BI Before induction

I2 2 hours after induction

I4 4 hours after induction

I6 6 hours after induction

I8 8 hours after induction

DESCRIPTION OF THE INVENTION

D-alanine is a essential component in the biosynthesis of the bacterial peptidoglycan in the cell wall of gram-positive and gram-negative bacteria (Wasserman, S. A., Daub, E., Grisafi, P., Botstein, D., and Walsh, C. T. (1984) Biochemistry 23, 5182-5187). D-alanine is generally present as a dipeptide in the C-terminal position of the UDP-N-acetylmuramyl- peptapeptide precursor of the peptidoglycan and is directly involved in crosslinking of adjacent peptidoglycan strands in the cell wall. The enzymes responsible for D-alanine are the alanine racemases (EC 5.1.1.1 ) that catalyzes the reversible racemisation between L- and D- alanine. Alanine racemases belong to the large and diverse group of PLP (vitamin B6) dependent enzymes. Although low sequence homology may be observed between groups of PLP enzymes, they may share the same common fold comprising PLP and similar catalytic mechanisms, which potentially results in limitations in substrate specificity. The majority of bacterial species normally possess either one or two distinct alanine racemase genes. The air gene encodes the constitutively expressed biosynthetic alanine racemase, which provides sufficient D-alanine for cell wall biosynthesis. The second gene encodes the so-called cata- bolic alanine racemase which is essential for /.-alanine catabolism. D-alanine auxotrophic

Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Listeria monocytogenes, and Lactobacillus plantarum strains have been generated by inactivating genes encoding alanine racemases. In Bacillus subtilis, a single alanine racemase gene (dal) is involved in alanine conversion. The presence of dal gene on a plasmid provides strong selective pressure for maintenance of the plasmid in chromosomal dal mutant Bacillus subtilis when grown on rich medium, but the complete genome sequence of B. subtilis confirms the existence of a second gene whose product shows high homology to alanine racemases. In the lactic acid bacteria (LAB) Lactobacillus plantarum, alanine racemase activity is encoded by homologous air gene, which if disrupted resulted in auxotrophy for D-alanine on rich media. In Listeria monocytogenes two genes that control the synthesis of the D-alanine, an alanine racemase gene (dal) and a D-amino acid aminotransferase gene (dat). By inactivaitng both genes, a L. monocytogenes strain growing only with the supplementation of D-alanine in culture or complementation of alanine racemase on plasmid was created. In Escherichia coli, two alanine racemases were identified. The air encoded alanine racemase is constitutively expressed, whereas the dadXencoded enzyme is essential only for /.-alanine catabolism, providing a substrate for a D-alanine specific dehydrogenase encoded b y the dadA gene. The dadX and dadA genes constitute an operon positively regulated by /.-alanine and repressed by glucose. The dadX gene product is the major source of alanine racemase activity (85% of the total activity) and is probably a secondary source of D- alanine for cell wall biosynthesis. Only the alr^'/dadX double mutant is dependent on D- alanine for growth. Removal of D-alanine from liquid rich medium during growth of an alanine racemase deficient strain resulted in rapid cell lysis. (Wijsman, H. J. (1972) Genet. Res. 20, 269-277). By analysis of isolated D-alanine prototrophic revertants of alr^'/dadX double knockout strain, it was surprisingly revealed that the revertant phenotype was linked to impairment of the L-methionine repressor MetJ and thus up-regulation of met genes among which cystathionine beta-lyase (MetC) was shown to have alanine racemase activity. The MetC alanine racemase coactivity and relation to the methionine pathway is depicted in Fig 1. In one embodiment of the present invention we have utilized an alr^'/dadX D-alanine auxotrophic strain for regulation of the integrity of the cell wall structure by L-methionine dependent expression of beta-cystathione lyase (MetC), which we have determined has alanine racemase activity. The alanine racemase knock-out strain was obtained by sequential deletion of air and dadX genes using the λ-red recombinase mediated recombination essentially as described previously ( Wasserman, S. A., Daub, E., Grisafi, P., Botstein, D., and Walsh, C. T. (1984) Biochemistry 23, 5182-5187, and Hols, P., Defrenne, C, Ferain, T., Derzelle, S., Delplace, B., and Delcour, J (1997) J. Bacterid. 179, 3804-3807) and proved to be D-alanine auxotrophic when grown on rich medium, but not on minimal medium without L-methionine. When the double knock-out strain was cultivated on LB medium supplemented with D-alanine for several generations, D-alanine prototrophic revertants could be isolated with low frequency. Upon proteome and transcriptome investigation of the revertants it was observed, that a general up-regulation of expression of genes involved in the methionine synthesis was detected in the revertant strain. The revertant phenotype was linked to mutations specifically occurring in the Met repressor. The genes involved in the biosynthesis of /.-methionine from homoserine and tetrahydrofolate are located at different positions on the E. coli chromosome and are under regulation by three different major factors. These include: i) repression of the Met genes by MeU aporepressor and it's corepressor S-adenosyl methionine (SAM), when E. coli is cultivated in the presence of Methionine; ii) activation of specific Met genes mediated by the MetR transcriptional activator and it's coactivator homocysteine and iii) repression of specific Met genes by vitamin B12. Mutations impairing metJ repressor function results in constitutive expression of the Met genes and decreased sensitivity to intracellular levels of L- methionine. (Nakamori. S, Kobayashi. S , Nishimura.T and Takagi.H (1999) Appl Microbiol Biotechnol 52:179-185).

According to the invention it was determined that E. coli cystathione-betalyase (MetC) is the protein responsible for alanine racemase coactivity from one of the up- regulated proteins as an expression plasmid encoding MetC was able to complement D- alanine auxotrophy and purified MetC showed a significant D-alanine racemase activity

The regulation of the integrity of the cell wall structure by L-methionine dependent MetC regulation was demonstrated using alkaline phosphatase as a recombinant model protein because alkaline phosphatase has an activity that can easily be measured both in growth media and subcellular fractions of the cell. Alkaline phosphatase is normally translo- cated to the periplasm and is thus a reporter for the amount of protein released by leakage from the cell wall to the growth medium from the periplasm after addition of L-methionine. When the gene Alkaline phosphatase (phoA) was induced by IPTG in KM5 defined medium without L-methionine, leakage of recombinant proteins from the periplasm of the host cell BL21 (DE3)/ alfdadX was be triggered by addition of L-methionine to the culture medium at early log phase of cell growth. When induction of protein synthesis was performed 1 hr later than initial L-methionine addition followed by feeding with L-methionine with three or four hours interval, significant amounts of alkaline phosphatase were expressed and secreted into growth medium compared to the cytoplasmic or periplasmic alkaline phosphatase. When air /dadX cells were incubated with low concentrations of L-methionine (eg. 0.01 mM-0.2mM) a significant inhibition is obserevd during exponential phase. However, at these concentrations the OD600 values reach approximately the same level or more as the controls without methionine by the end of the overnight cultivation. This also indicates that the alf/dadX cells are both viable after treatment with these concentrations and also capable of secreting alkaline phosphatase to the growth medium.

It should be clear to the person skilled in the art that the regulation of the integrity of BL21 (DE3) alrdadXceW wall structure and thus leakage of recombinant target protein to the medium by L-methionine can be optimized using variations of the disclosed embodiments of the invention by balancing the amount of alanine racemase activity during growth without de- straying the cells ability to respond to L-methionine in a way that increases the obtainable cell density. Balancing of the amount of alanine racemase activity present in the cell can be obtained by mutating the methionine repressor, the MetC gene or operator sequence region, through MetC operon modified regulation of an inserted air gene.

The concept of regulating the integrity of BL21 (DE3) alrdadXceW wall structure and thus leakage of recombinant target protein to a minimal medium (without methionine) by L- methionine can be proved using variations of the above example. Non-limiting examples of the invention include a double knock out alr-dadX E.coli strain

i) with one or more copies of the wildtype MetC gene and its operator region on a low copy plasmid and one copy of wildtype metJ on chromosome; ii) with one or more copies of the MetC gene and its operator region integrated into the genome at the air or alr/dadX chromosomal position iii) with one copy of wildtype metC and one copy of mutant metJ on chromosome and iv) with one copy of a gene cassette comprising the air gene under the control of a metC operator region that is optimized by mutagenesis to be partly responsive to MetJ binding integrated into the genome at the air or alr/dadX chromosomal position.

The growth inhibitory effect of L-methionine was demonstrated for i) and iii), which was characterized by having a shorter lag phase than observed for the unmodified BL21 (DE3) alr^'dadX. The host cells in i) and iii) may achieve higher OD values in the first hours of cultivation, but needs higher amounts of L-methionine to repress the MetC alanine racemase activity due to the presence of extrachromosomal MetC on a plasmid and due to a partly functional methionine repressor, which is less responsive to intracellular levels of L- methionine.

A partly functional Met J repressor may be obtained by point mutations or truncations in the MetJ protein, which interferes with MetJ dimerization, binding to the corepressor SAM, or the interaction with the Met Box. Non-limiting examples are MetJ(Leu36Thr), MetJ(Arg42Cys), MeU(GIyI 5Ser), MeU(AIaI 2Thr), MeU(Ala60Thr) and MeU(His50Asn). A short cultivation time may be advantageous for industrial scale production using the method according to the present invention.

As the growth of the BL21 (DE3) alr^'dadX strain is significantly inhibited during the exponential phase the other alternatives may be used for more rapid fermentations as they allow leakage to the growth medium at late exponential or immediately after that phase, when higher concentration of L-methionine is added.

The invention also includes using functional analogues of L-methionine such as D- ethionine, which may be more stable and less likely catabolized than L-methionine and thus be advantagoues to use instead of L-methionine for the regulated expression of MetC and balancing of alanine racemase activity. Other functional analogues of L-methionine are cis- crotylglycine, 2-aminoheptanoic acid, norvaline, 2-butynylglycine, DL-norleucine and allylgly- cine.

The invention also includes other applications of MetC in relation to controlled leakage from alanine racemase deficient strains in which no L-methionine needs to be added. An example includes using BL21 (DE3) alr^'dadX cells in which the genomic wt MeU gene se- quence and its operator/promoter region is completely replaced with a wt MeU or mutated partly functional variants of MeU under the tight control of a T7 promoter with a lacl operator region. This strain will as the revertant strain described above be able to grow as expression of Met genes would be constitutive and MetC would provide D-alanine for maintaining cell wall integrety. When protein induction from a plasmid encoding target protein is induced by IPTG, MeU would also be induced, which will repress the methionine genes including MetC and cause leakage of the cell wall.

The invention is applicable to all bacterial host cells, for which alanine racemase genes can be disrupted by state of the art knock-out technology and which has a cystathionine beta-lyase gene which encodes a protein with a regulation and function that is similar to MetC from E. coli. Due to the large functional overlap between PLP (pyrodoxal phosphate) dependent enzymes the other candidates from the methionine pathway may have alanine racemase activity in other organisms. Non-limiting examples are PLP dependent enzymes, which has documented methionine regulation in E. coli, such as tryptophan synthetase (TrpB), hypothetical protein YbdL, Cystathionine gamma synthetase (MetB), Glycine serine dehydrogenase (GIyA) and aspartate semialdehyde dehydrogenase (Asd).

The advantage of periplasmic expression followed by regulated secretion of recombinant proteins to the culture medium of E. coli includes less contamination of product by host proteins, reduced proteolytic activity, enhanced protein folding capacity, higher product stability and solubility, less dependence on mechanical steps after expression and thus less manufacturing expenses.

The present invention is applicable to all recombinant proteins of interest which can be targeted to the periplasm of E. coli. The recombinant protein of interest may be expressed from state of the art E. coli plasmids or from copies of the recombinant gene inserted in the E. coli genome. No limiting examples of expression plasmids are pET, pACYCs pBAD, pTrc (Lee, N., Francklyn.C. and Hamilton, E.P. (1987).PiOC.Natl.Acad.Sci.USA 84, 8814-8818)

The recombinant protein is preferably targeted to the periplasm by means of an N- terminal signal peptide sequence such as phoA, degQ, degS, degP, OmpA, OmpF, OmpH, OmpP, OmpT, lamb or pelB (from Erwania carotovora) or variants thereof which have been optimized for more efficient signal peptidase cleavage in the inner membrane and thus the efficiency of translocation across the inner membrane.

Non limiting examples of recombinant target proteins relevant for this invention are: aprotinin, tissue factor pathway inhibitor or other protease inhibitors, insulin, insulin analogues or insulin precursors, human or bovine growth hormone, interleukin, glucagon, GLP-1 , GLP-2, IGF-I, IGF-II, tissue plasminogen activator, transforming growth factor α or β, platelet-derived growth factor, GRF (growth hormone releasing factor), immunoglubolines, EPO, TPA, protein C, blood coagulation factors such as FVII, FVIII, FIV and FXIII, exendin-3, exentidin^, and enzymes or functional analogues thereof.

Other examples of target proteins are transforming growth factor α (TGF-α), transforming growth factor β (TGF-β), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), thrombopoietin (TPO), interferon, pro-urokinase, urokinase, plasminogen activator inhibitor 1 , plasminogen activator inhibitor 2, von Willebrandt factor, a cytokine, e.g. an interleukin such as interleukin (IL) 1 , IL-I Ra, IL-2, IL-4, IL-5, IL-6, IL-9, IL-1 1 , IL-12, IL-13, IL- 15, IL-16, IL-17, IL-18, IL-20 or IL-21 , a colony stimulating factor (CFS) such as GM-CSF, stem cell factor, a tumor necrosis factor such as TNF-α, lymphotoxin-α, lymphotoxin-β, CD40L, or CD30L, a protease inhibitor e.g. aprotinin, an enzyme such as superoxide dismu- tase, asparaginase, arginase, arginine deaminase, adenosine deaminase, ribonuclease, catalase, uricase, bilirubin oxidase, trypsin, papain, alkaline phosphatase, β-glucoronidase, purine nucleoside phosphorylase or batroxobin, an opioid, e.g. endorphins, enkephalins or non-natural opioids, a hormone or neuropeptide, e.g. calcitonin, glucagon, gastrins, adreno- corticotropic hormone (ACTH), cholecystokinins, lutenizing hormone, gonadotropin- releassing hormone, chorionic gonadotropin, corticotrophin-releasing factor, vasopressin, oxytocin, antidiuretic hormones, thyroid-stimulating hormone, thyrotropin-releasing hormone, relaxin, prolactin, peptide YY, neuropeptide Y, pancreastic polypeptide, leptin, CART (cocaine and amphetamine regulated transcript), a CART related peptide, perilipin, melano- cortins (melanocyte-stimulating hormones) such as MC-4, melanin-concentrating hormones, natriuretic peptides, adrenomedullin, endothelin, secretin, amylin, vasoactive intestinal peptide (VIP), pituary adenylate cyclase activating polypeptide (PACAP), bombesin, bombesin- like peptides, thymosin, heparin-binding protein, soluble CD4, hypothalmic releasing facto- rand melanotonins or functional analogs thereof. In another embodiment of the invention the target protein may be a processing enzyme such as proteases (eg enterokinase, caspases trypsine like serine proteases), lipase, phospatase, glycosyl hydrolases (eg. mannosidases, xylosidases, fucosidases), kinase, mono or dioxidase, peroxidase, transglutaminase, car- boxypeptidase, amidase, esterase, and phosphatase.

In another embodiment of the invention the target protein may be recombinant anti- body fragments which are either monovalent (Fab.scFv, single variable VH and Vl domains) or bivalent fragments (Fab'2, diabodies, minibodies, Bis-scFv etc) used for therapeutic or diagnostic purposes.

The expression of functional antibody fragments in E.co/i has been accomplished through secretion the light chain and heavy chain fragments into the periplasm separately, where proper assembly and disulfide bond formation can take place. Bivalent F(ab')2 fragment are necessary for clinical applications, which can provide longer circulating half time in patients than Fab' fragment does. Antibody fragments are prone to be degraded and form inclusion bodies when expressed in periplasm. If the antibody fragment can be secreted further into medium once it is translocated into periplasm the above problems could be dimin- ished.

With relation to expression of recombinant antibodies, the invention can also be used to improve existing methods for screening and isolation of high-affinity antibodies.

Anchored periplasmic expression (APEX) is a technology for the screening and isolation of ligand-binding proteins form combinatorial libraries anchored on the periplasmic face of the inner membrane of E.coli (Barrett R. H. et.al 2004). In this strategy, proteins are trans- located to the periplasm, targeted and bound to the inner membrane of E.coli via lipidation of a small N-terminal 6-aa fusion. After chemical/enzymatic permeabilization of the bacterial outer membrane, E.coli expressing anchored scFv antibodies can be specifically labelled with fluorescent antigens. After a washing step, cells that express both the fluorescent anti- gen and an APEX-anchored scFv are highly fluorescent and can be readily sorted from cells that express either only an scFv or GFP-antigen fusion alone using flow cytometry. After sorting the target DNA can be recovered by PCR and further cloning step. When combining the BL21 (DE3) alfdadX and the strategy outlined by the present invention with the APEX or related methods, no extra step for treatment of cell with EDTA or lysozyme to increase the permeability of the bacterial outer membrane is needed. L-methionine is instead used to regulate the permeability of the cell membrane, which is much milder than treatment with EDTA and lysozyme and which will allow the cells to be viable enough for growth after sorting, so that no additional PCR and cloning steps are needed.

The nucleic acid construct used in the present invention may suitably be of genomic or cDNA origin, for instance obtained by preparing a genomic or cDNA library and screening for DNA sequences coding for all or part of the fusion protein by hybridization using synthetic oligonucleotide probes in accordance with standard techniques ( Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd. Ed. Cold Spring Harbor Labora-tory, Cold Spring Harbor, New York, 1989). The nucleic acid construct used in the present invention may also be prepared synthetically by established standard methods, e.g. the phosphoamidite method described by Beaucage and Caruthers, Tetrahedron Letters 22 (1981 ), 1859 - 1869, or the method described by Matthes et al., EMBO Journal 3 (1984), 801 - 805. According to the phosphoamidite method, oligonucleotides are synthesized, e.g. in an automatic DNA synthesiser, purified, annealed, ligated and cloned in suitable vectors. The DNA sequences encoding the fusion protein may also be prepared by polymerase chain reaction such as splicing by overlap extension PCR using specific primers, for instance as described in US 4,683,202, Saiki et al., Science 239 (1988), 487 - 491 , or Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, 2nd. Ed. Cold Spring Harbor Labora-tory, Cold Spring Harbor, New York,. Furthermore, the nucleic acid construct may be of mixed synthetic and genomic, mixed synthetic and cDNA or mixed genomic and cDNA origin prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate), the fragments corresponding to various parts of the entire nucleic acid construct, in accordance with standard techniques. The DNA sequences used in the present invention are usually inserted into a re- combinant vector which may be any vector, which may conveniently be subjected to recom- binant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e. a vector, which exists as an extra chromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.

The vector is preferably an expression vector in which the DNA sequence used in the present invention is operably linked to additional segments required for transcription of the DNA. In general, the expression vector is derived from plasmid or viral DNA, or may con- tain elements of both. The term "operably linked" indicates that the segments are arranged so that they function in concert for their intended purposes, e.g. transcription initiates in a promoter and proceeds through the DNA sequence coding for the fusion protein.

Expression vectors for use in expressing the target protein will comprise a promoter capable of directing the transcription of a cloned gene or cDNA. The promoter may be any DNA sequence, which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.

Examples of suitable promoters for use in bacterial host cells include the promoter of the Bacillus stearothermophilus maltogenic amylase gene, the Bacillus licheniformis alpha- amylase gene, the Bacillus amyloliquefaciens BAN amylase gene, the Bacillus subtilis alka- line protease gen, or the Bacillus pumilus xylosidase gene, or the phage Lambda P_R or P_L promoters or promoters used for expression in E. coli eg. lac, trp, phoA, araBAD, tac, bacteriophage T7 and cspA.

The vector may also comprise a selectable marker, e.g. a gene product which complements a defect in the host cell, such as the gene coding for dihydrofolate reductase (DHFR) or a marker gene which confers resistance to a drug, e.g. ampicillin, kanamycin, tet- racyclin, chloramphenicol, neomycin, hygromycin or methotrexate

Examples of bacterial host cells which, on cultivation, are capable of producing the target protein are grampositive bacteria such as strains of Bacillus, such as strains of B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliq- uefaciens, B. coagulans, B. circulans, B. lautus, B. megatherium or B. thuringiensis, or strains of Streptomyces, such as S. lividans or S. murinus, or gramnegative bacteria such as strains of Echerichia coli. The transformation of the bacteria may be effected by protoplast transformation or by using competent cells in a manner known per se (cf. Sambrook et al., supra). The transformed or transfected host cell is then cultured in a suitable nutrient medium under conditions permitting expression of the fusion protein after which all or part of the resulting peptide may be recovered from the culture. The medium used to culture the cells may be any conventional medium suitable for growing the host cells, such as minimal or complex media containing appropriate supplements. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. in catalogues of the American Type Culture Collection).

In the present context the three-letter or one-letter indications of the amino acids have been used in their conventional meaning as follows: Glycine (GIy & G), proline (Pro & P), alanine (Ala & A), valine (VaI & V), leucine (Leu & L), isoleucine (lie & I), methionine (Met & M), cysteine (Cys & C), phenylalanine (Phe & F), tyrosine (Tyr & Y ), tryptophan (Trp & W), histidine (His & H), lysine (Lys & K), arginine (Arg & R), glutamine (GIn & Q), asparagine (Asn & N), glutamic acid (GIu & E), aspartic acid (Asp & D), serine (Ser & S) and threonine (Thr & T). If, due to typing errors, there are deviations from the commonly used codes, the commonly used codes apply.

Unless indicated explicitly, the amino acids mentioned herein are L-amino acids. Further, the left and right ends of an amino acid sequence of a peptide are, respectively, the N- and C-termini unless otherwise specified.

All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference in their entirety and to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein (to the maximum extent permitted by law).

All headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way. The use of any and all examples, or exemplary language (e.g. "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. The citation and incorporation of patent documents herein is done for convenience only and does not reflect any view of the validity, patentability, and/or enforceability of such patent documents.

This invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. EXAMPLES

Example 1

Construction of an alfldadX E. coli strain

An alr/dadX deficient strain was accomplished by sequential deletion of a/rand dadX genes using the λ-red recombinase mediated recombination as described previously (Datsenko, K. A. and Wanner, B. L. (2000) Proc Natl Acad Sci U S A 97,6640-6645;2 and Murphy, K. C, Campellone, K. G., and Poteete, A. R. (2000) Gene 246, 321-330 ). Oligos used for the generation of recombinant fragments are shown in Table 1 and were synthesized by TaKaRa Biotechnology (Dalian) Co., Ltd. To construct an alr^~ strain, 5' flanking se- quence (0.52 kb) of air was PCR amplified from the chromosomal DNA of strain BL21 (DE3) with primers FaIrL and RaIrL. The PCR product {alrL) was cloned into pGEM-T vector, resulting in pGEM-a/rL. 3' flanking sequence (0.55 kb) of a/rwas PCR amplified with primers FaIrR and RaIrR. The PCR product (alrR) was also cloned in a commercially available plasmid pGEM-T vector resulting in pGEM-a/rR. alrR fragment was released from pGEM-a/rR with Hindi 11 and BamHI digestion and cloned between corresponding sites of pGEM-a/rL, resulting in pGEM-Δa/r Kanamycin-resistant gene flanked by FRT (FIp recombinase target) sites was amplified by two runs of PCR with pET39b as template and primers Fkan/Rkan and FaFRT/RaFRT. The final amplican was inserted at Hind 111 site of pGEM-Δa/r, creating the air replacement vector pGEM-Δa/r/FRT-Km. To construct a dadX strain, 5' flanking sequence (0.73kb) of dadX was PCR amplified from the chromosomal DNA of strain BL21 (DE3) (US patent 4,952,496) with primers FdadXL and RdadXL. The PCR product (dadXL) was cloned in pGEM-T vector, resulting in pGEM- dadXL. 3' flanking sequence (0.73 kb) of dadX was PCR amplified with primers FdadXR and RdadXR. The PCR product (dadXR) was also cloned in pGEM-T vector, resulting in pGEMT- dadXR. dadXR fragment was released from pGEM-dadXR with Ndel and Sacl digestion and cloned between corresponding sites of pGEM-dadXL, resulting in pGEM-ΔdadX. Kanamycin- resistant gene flanked by FRT sites was amplified with primers FdFRT/RdFRT and pGEM- Δa/r/FRT-Km as template. The PCR product was inserted between pstl and Ndel sites of pGEM-ΔdadX, creating the dadX replacement vector pGEM-ΔdadX/FRT-Km. BL21 (DE3) cells carrying a Red helper plasmid (pKD46) (£. coli Genetic Stock Center, Yale University) were grown in 50 ml LBA liquid at 30°C with 300 rpm rotation until OD600 reached to 0.4. 1 mM L-arabinose was added to cell culture at 30°C for about an hour until OD600 reached 0.6. Then the cells were made electro-competent by concentrating 100 fold and washing three times with ice-cold 10% glycerol. The replacement DNA frag- merits containing a kanamycin resistance gene flanked on both sides by homologous sequences of target genes were prepared by digestion of replacement vector with corresponding enzymes, gel-purified, and suspended in water. Electroporation was done by using Mi- cropulser (Biorad) with 2.5 Kv voltage booster and 0.2-cm chamber according to the manu- facture's instruction by using 40 μl of cells and 100ng of replacement fragment. The shocked cells were added to 1-ml SOC, incubated 1 hr at 37°C, and then spread onto LB agar with kanamycin (50 μg/ml) and D-alanine (100 μM) to select KmR transformants. KmR resistant colonies were screened by PCR with primers annealing to regions outside of the mutated gene. Next, the antibiotic gene was excised by introducing the plasmid pCP20 encoding the FLP recombinase (£. coli Genetic Stock Center, Yale University). Plasmids pKD46 and pCP20 are both thermosensitive for replication and were cured at 42°C(1 ;2).

The BL21 (DE3)a/r/dadX strain displayed a strict dependence on the presence of D- alanine on rich LB medium, suggesting that disruption of both a/r and dadX genes resulted in a D-alanine auxotrophic phenotype.

Growth characteristics of BL21(DE3)alrdadX strain grown in defined medium when different amounts of L-methionine are added during cultivation.

To obtain the defined KM5 growth medium (without methionine) for cultivation of BL21 (DE3) alr^'dadX cells the following components were added to one liter ion free water; 1.7g Citric acid (monohydrate), 8 g (NhU)₂HPO₄, 9.2 g KH₂PO₄, 1 ml KM5 trace metal solution, pH is adjusted to 6.3 with 5N NaOH. After sterilizaition the following was added: 105 mg FeSO₄.7H₂O(by filtration), 4.9 ml 1 M MgSO₄,7H2O, 0.2 ml 0.1 M CaCI₂, 33 ml 50% (w/L) glucose.

Figure 2 shows the growth of the BL21 (DE3) alr^'dadX strain in KM5 medium upon addition of different amounts of L-methionine to the growth medium at the time of inoculation Growth of cell BL21 (DE3) alr^'dadX can be significantly inhibited by L-Met even in very low concentration at 0.1 mM during the exponential phase of growth. Importantly, when concentrations from 0.1 mM to 1 mM are used the growth inhibitory effect is reversed and the OD600 values reach the same level or more as the control without methionine by the end of the overnight cultivation. This also indicates that the cells are viable after treatment with these concentrations. However, when 1 OmM L-Methionine were added, the growth of the cell is totally inhibited and undergo cell lysis. The clear indication that growth is inhibited by addition of L-methionine during the exponential phase is in agreement with the assumption that, L- Met will affect the synthesis of cell wall because of the inefficient production of D-Alanine for peptidoglycan synthesis.

Ultra-structure analysis of the effect of L-methionine on the formation of cell wall To confirm that addition of L-methionine directly affects the integrity of the outer membrane, electron microscopy was performed on BL21 (DE3)alr/dadx grown on defined medium with or without L-methionine. Colonies of BL21 (DE3)alr/dadx from overnight culture plates were inoculated at OD 0.3 into CM5 defined medium without L-methionine at 37oC. When OD reached 1 , L-methionine was added to the culture at final concentration of 0, 0.01 , 0.1 and 1.0 mM, and growth was continued at 3O⁰C for 6 hrs. For transmission electron microscopy (TEM) cells were harvested by low speed centrifugation (1000g, 5 min), washed twice in PBS buffer (pH 7.4), pre-fixed in 3% (w/v) glutaraldehyde and fixed with 1 % (w/v) osmium tetroxide (OsO₄₎. After dehydration with increasing concentrations of ethanol, cells were embedded with EPOX 812 and polymerized at 60 °C for 48 h. To improve contrast, the samples were stained with 4% (w/v) uranyl acetate and then with 0.4% (w/v) lead citrate. Ul- trathin sections were deposited on a copper grid and viewed in a PHILIPS CM120 electron microscope at 120 kV using a 30-mm objective aperture.

When 0.01 mM L-Met was added into growth medium, the majority of cells showed normal morphology, but for some cells the cell wall formation was inhibited and cells dis- played a spherical morphology and blebbing of the outer membrane compared to the controls without methionine. This morphological state is similar to what is observed when peptidoglycan disrupting antibiotics, such as penicillin is added to E. coli. The data therefore confirms that the MetC activity, which provides D-alanine for crosslinking of the peptidoglycan, has been inhibited by addition of L-methionine to the cells. When the concentration of L- methionine was increased to 0.1 mM most cells showed this atypical morphology. When very high concentrations (e.g. 1 mM) of L-methionine were added a significant increase in the amount of dead cells was observed.

Example 2 The MetC gene expression cassette under regulation of its own promoter, which is responsive to MeU, was cloned into pACYC and pETduet vectors (Novagen) using standard cloning protocols. Both expression of MetC from pACYC-MetC-OP and pETduet-MetC-OP provided enough alanine racemace to support more robust growth of the BL21 (DE3)/ air dadX cell in defined medium. The growth rate of pACYC-MetC-OP/β/^ ?(DE3ja/r-dadx- and pETduet-MetC-OP/S/.27(OE3,) alfdadX in the early and middle log phase is faster than the one of BL21 (DE3)/alr-dadX- without plasmid in defined medium. MetC on the high copy number plasmid presumably can not be effectively controlled by the endogenous amount of MetJ product and SAM present in the cell as no growth inhibitory effect was obtained by addition of L-methionine. However, with the low copy number plasmid pACYCduet-MetC-OP, the inhibition effect of L-Met to the cell growth was observed with the similar pattern as the BL21 (DE3) alfdadX cell. Thus, expression of MetC from low copy plasmids can be used to support the growth of the cell more rapidly and keep the regulatory activity of MetC gene as well. This results shows that it is feasible to fine tune alanine racemase activity to balance the optimal growth of cell and regulatory secretion by L-Met Another way of balancing alanine racemase activity for optimal growth as well as regulation efficiency of secretion was to utilize the mutations in MetJ in the BL21 (DE3)a/r dadX cell. Mutated MetJ partially impairs repression of MetC which results in a small increase in the intracellular levels of alanine racemase activity delivered by MetC, thus allowing higher growth rate. In general, low levels of L-methionine used to inhibit the growth of BL21 (DE3)a/r dadX did not have an significant effect on the strains with MetJ mutations. In contrast the MetJ mutated strains could withstand much higher concentrations of L-methionine. This is in agreement with the impaired MetJ activity of these cells. MetJ mutated cells were divided into two groups on the basis of their growth response to L-Met with or without 2 mM or 10 mM L- methionine treatment. Group I included MetJ Arg42Cys, MetJ1-45aa(truncated) and Met- JAIaδOThr, which could only be significantly inhibited by high level of L-Met (10 mM). These mutations might significantly decrease MetJ ability to dimerize and interact with L-Met or SAM. Group Il includes MetJHisδOAsn, MetJGIy15Ser, MetJLeu36Phe and MetJAIa12Thr, which could be significantly inhibited by low level of L-Met (2 mM).

Example 3

Secretion expression of phoA gene by the system BL21 (DE3)aJ 'rdadXwith L-Met induction.

To monitor the expression and secretion of a recombinant protein in the BL21 (DE3)a/rdad>Chost cells during induction with IPTG and addition of L-methinonine, clon- ing of a pET plasmid comprising Alkaline phosphatase(PhoA) was performed:

PhoA gene was amplified by standard PCR methods from genomic DNA of strain BL21 (DE3)a/rdadX by using primers phoA-1 and phoA-2. The PCR amplification was carried out by initial denaturation at 95°C/30 sec followed by 20 cycles of 95°C/30sec (denaturation), 55°C/ 60 sec (annealing), and 72°C/60 sec (elongation), final extension was performed at 72°C for 10 min by Pfu from Invitrogen according to instruction of the manufacturer. The PCR fragment was ligated into vector pET39b(Novagen) from site Ndel and Kpnl, to generate expression plasmid pET39b-phoA (Fig 3). Two primers phoA-3 and phoA-4 were designed for change mutagenesis of phoA into phoAm by changing D101 S, which reported have 30 fold improvement for enzyme activity (Brickman. E and Beckwith.J,J.Mol.Biol.(1975), 96, 307-316) thus making the enzyme assay more sensitive.

Overnight culture of BL21 (DE3)a/r dadX or wildtype BL21 (DE3) controls transformed with pET39b/phoA were prepared from LB amp plates for BL21 (DE3) dadX/alr or LB+amp for wild type strain cultivated O/N at 37 degrees C. 20ml KM5 defined medium was inoculated to reach a start OD 600 of 0.3. L-methionine was added at final concentration of 0.05mM,

0.1 mM and 0.2mM at early-Log phase (OD600=1.0). After culturing for additionally one hour at 37°C, the temperature was lowered to 30⁰C and induction of alkaline phosphatase was initiated at with 0.05 mM IPTG. After induction and during the rest of the cultivation, additional L-methionine in the same concentration as the initial addition was added at interval of 3hrs or 4hrs. After overnight induction cells were harvested and growth medium fractions were prepared after centrifugation at 12,000rpm for 10 min at room temperature. Periplasmic fractions were prepared according to the standard Protocol of PeriPreps™ Periplasting Kit (EPICENTRE). After dilution of samples, 10ul was used for alkaline phosphatase activity measurement using a method essential as described by Edith Brickman (Glaser SM, Yelton DE and Huse WD, J. Immunology (1992), 149, 3903-3913). The assay plates were read at OD420 by FLuostar Optimal spectrophotometer and activity units of phosphatase were calculated by the following formula: 1000^χOD420^χdilution factor / time *OD600.

D-Alanine is required during the period of exponential growth when the cells actively divide to sustain the cell wall integrity. 0.01 mM, 0.05 mM and 0.1 mM L-methionine added in intervals during overnight culturing does not have a significant effect on the secreted amount of protein as the level of phoA activity measured in the growth medium is at the same level as the two controls constituting the wildtype BL21 (DE3)/pET39bphoA and BL21 (DE3)a/r dadX7pET39bphoA without addition of L-methionine. However, if a cone. 0.2 mM L- methionine is added with 3 or 4 hour intervals during the overnight culturing a significant amount of alkaline phosphatase is leaked into the growth medium compared to the controls, indicating that a constant critical level of intracellular L-methionine is required to facilitate repression of MetC gene and secretion of periplasmic phoA to the growth medium. Fig 4 shows that the majority of the alkaline phosphatase is released from the periplasm with 0.2 mM L- methionine as most of the activity was detected in the growth medium. A slight decrease in the cell density is observed at this condition. However, taken together with the results presented in example 2 it can be concluded that a sustainable method for regulated leakage of recombinant protein from the periplasm can be achieved using optimized and constant concentrations of added L-methionine.

Table 1

Example 4

Evaluation of Ly49-5E6 F(ab')2-LZ expression in cell BL21 (DE3) alr-::MetCOP dadx- by feed batch fermentation The expression of functional antibody fragments in E.coli has been accomplished through secretion the light chain and heavy chain fragments into the periplasm separately, where proper assembly and disulfide bond formation can take place. Bivalent F(ab')2 fragments are necessary for clinical applications, which can provide longer circulating half time in patients than Fab' fragment does. Antibody fragment are prone to be degraded and form in- elusion bodies when expressed in periplasm. If the antibody fragment can be secreted further into medium once it is translocated into periplasm, the above problems could be diminished. BL21 (DE3)alr-::MetCOPdadX- was tested for regulatory secretion of Ly49-5E6 F(ab')2-LZ (Ref: Sentman,C.L,Hanckett,J., Kumar, V.and Bennett,M,1989). H-2/Hh-1 specific subsets of murine natural killer cells mediate rejection of bone marrow grafts, J. Exp. Med.170, 191-202) in 5 L fermentor. The KM5 medium used in fermentation was essentially the same as described in Example 1 , except that 40 mM magnesium sulphate and 1 g L glucose was used.

The cultivation started at OD600 0.2, and continued for 16.5 hours at 37°C before performing

induction at 30°C for 8 hours. For the control strain BL21 (DE3), IPTG concentration for induction is 0.25mM. Three conditions were tested in BL21 (DE3)alr-::MetCOPdadX- cells as indicated on Fig. 5: 0.1 mM IPTG, 0.25mM IPTG and 0.25mM IPTG plus 1 mM of L-

Methionine, which was added at OD600 0.5. The expression level of Ly49-5E6 F(ab')2-LZ in both culture medium and periplasmic fraction were evaluated by western blot with an anti- mouse Fab antibody. In the periplasmic fraction, strain BL21 (DE3)alr-::MetCOPdadX- showed similar expression level of Ly49-5E6 F(ab')2-LZ as the control strain BL21 (DE3) (Fig. 6). However, in growth medium, BL21 (DE3)alr-::MetCOPdadX- showed much higher levels Ly49-5E6 F(ab')2-LZ compared to BL21 (DE3) controls. When comparing levels of secreted Ly49-5E6 F(ab')2-LZ in BL21 (DE3)alr-::MetCOPdadX- cells using either 0.25mM IPTG plus

1 mM L-Methionine or 0.25mM IPTG alone, about 5-fold more Ly49-5E6 F(ab')2-LZ was detected upon addition of L-Met (Fig. 6). Thus, the addition of 1 mM L-Met at the exponential phase promoted secretion of antibody fragment into medium and in turn enhanced the overall expression level.

Claims

1. Method for extracellular production of proteins in alanine racemase deficient bacteria strains wherein leakage of the expressed target protein to the culture medium through the cell wall membrane is triggered by repression of the cystathionine beta-lyase (MetC) encod- ing gene.

2. Method according to claim 1 , wherein the repression of the cystathionine beta-lyase encoding gene is regulated by addition of L-methionine or an analogue thereof to the culture medium after an initial growth phase.

3. Method according to 2, wherein L-methionine or a functional analogue thereof is added when a high cell density has been obtained.

4. Method according to claims 1-3, wherein the host cell is transformed with a suitable plas- mid expressing cystathionine beta-lyase under regulation of its own operator region.

5. Method according to claims 1-3, wherein at least one copy of cystathionine beta-lyase encoding DNA under regulation of the cystathionine beta-lyase operator region is integrated into the chromosome of the host cell in the original position of the air or alr/dadx gene.

6. Method according to claims 1-3, wherein the air gene or the dadX gene under the control of an at least partly functioning cystathionine beta-lyase operator region is integrated into the host cell genome.

7. Method according to claim 1 , wherein the host cell carries a mutation in the MetJ gene rendering MetJ partially functioning.

8. Method according to claim 1-7 wherein L-methionine or an analogue thereof is added in an amounts of from about 0.1 to about 5 mM at intervals of 1-20 hours.

9. Method according to claim 8, wherein L-methionine or an analogue thereof is added in an amounts of from about 0.1 to about 2 mM at intervals of 2-5 hours.

10. Method according to claim 1 , wherein the MetJ gene is under regulation by the same promoter/operator which is used for the target protein.

11. Method according to claim 10, wherein the MetJ gene and the DNA coding for the target protein are both under control of the T7/lacl promoter.

12. Method according to any of the previous claims, wherein the host cell posses a double knockout of the alanine racemase genes.

13. Method according to claim 12, wherein the host cell is an alr/dadX double knock out strain.

14. Method according to any of the previous claims wherein the cell is an E. coli strain.

15. Method according to any of the previous claims, wherein the target protein is a recombinant antibody fragment.