Expression of Ovine Gamma Interferon in Escherichia coli and ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1994, p. 1641-1645

Vol. 60, No. 5

0099-2240/94/$04.00+0 Copyright C) 1994, American Society for Microbiology

Expression of Ovine Gamma Interferon in Escherichia coli and Corynebacterium glutamicum H. BILLMAN-JACOBE,I* A. L. M. HODGSON,' M. LIGHTOWLERS,2 P. R. WOOD,' AND A. J. RADFORD1t

Commonwealth Scientific and Industrial Research Organisation, Division ofAnimal Health, Parkville, Victoria, 3052,1 Clinical Veterinary Centre, Melboume University, Werribee, Victoria, 3030,2 Australia Received 9 September 1993/Accepted 26 February 1994

Bacteria of two species, Escherichia coli and Corynebacterium glutamicum, were used as hosts to express recombinant ovine gamma interferon as a fusion protein with glutathione S-transferase. The recombinant gamma interferon produced by both bacteria was biologically active in vitro and was recognized by anti-gamma interferon monoclonal antibodies. E. coli produced large amounts of soluble recombinant protein which could be purified by a simple affinity chromatography method. Only a small fraction of the recombinant protein made by C. glutamicum was recovered by this method. Expression of recombinant protein in C. glutamicum was unstable but could be controlled by increased regulation of the tac promoter. Both hosts expressed ovine gamma interferon at high levels, with the recombinant protein making up a significant proportion of the cellular protein content.

When selecting an expression system for production of recombinant proteins, one cannot always predict how well the protein will be expressed in the selected host. Often, several systems need to be evaluated before the best combination of yield, solubility, purity, and biological activity is achieved. This paper expands the range of choices available by examining recombinant ovine gamma interferon (IFN--y) expression in Escherichia coli and in Corynebacterium glutamicum as an alternative bacterial host for recombinant expression. Human IFN--y is produced for pharmaceutical use, and a considerable effort has been made to express it in a variety of expression systems (4, 6). Large amounts of recombinant human IFN-y were produced by E. coli (13), but mostly in the form of insoluble inclusion bodies. Pure, soluble, and active IFN-y was obtained only after extensive treatment of the inclusion bodies. Ovine IFN--y has been expressed in a eucaryotic expression vector in Cos-1 monkey fibroblasts (5). Although biological activity was detected in the culture media, this expression system is limited by the small amounts of recombinant protein that can be produced and by the difficulty of purification. We evaluated the expression of recombinant ovine IFN--y in two bacterial expression systems. The first aim was to use pGEX expression vectors (19) in E. coli to make recombinant IFN-y which could be easily purified. Recombinant proteins expressed from pGEX vectors are translated as glutathione S-transferase (GST) fusion proteins which can be purified in a single step by affinity chromatography (19). Our second aim was to test the capacity of C. glutamicum for recombinant protein expression. C. glutamicum is a gram-positive organism that has been used industrially for several decades for amino acid synthesis (10). Some strains of C. glutamicum have been engineered to produce large quantities of amino acids such as lysine, which has been produced at levels up to 100 g/liter of culture (1). This huge metabolic capacity makes C. glutamicum

a strong candidate as a host for recombinant protein production. C. glutamicum is nonpathogenic and has no associated hazardous toxins, offering considerable advantages as a production strain.

MATERUILS AND METHODS Bacterial strains and molecular techniques. E. coli JM109 and C. glutamicum AS019 were used as the bacterial hosts for gene expression. DNA manipulations were carried out as described by Sambrook et al. (18). Restriction endonucleases and DNA modification enzymes were obtained from AMRADPharmacia and Promega. Construction of expression plasmids pGEXIFN and pPGIFN. The gene sequence encoding the mature protein of ovine IFN--y was subcloned from pUC18IFN (16) as an MscI-EcoRI fragment into Smal-EcoRI-digested pGEX-2T (19) to produce pGEXIFN for expression in E. coli. This plasmid was modified for use in C. glutamicum by fusion with a shuttle vector, pEP2 (15). The shuttle vector carries an origin of replication that functions in E. coli and corynebacteria and a kanamycin resistance marker. Both plasmids, pEP2 and pGEXIFN, were linearized by EcoRI digestion and ligated to produce pPGIFN. The orientations of GST and IFN were in the same direction as the kanamycin resistance gene on pEP2. Genetic transfer. Plasmids were introduced into both E. coli and C. glutamicum by electroporation by use of a Bio-Rad Gene Pulser with cuvettes with 0.2-cm electrode gaps. E. coli cells were prepared according to instructions from Bio-Rad. C. glutamicum cells were made electrocompetent (7), electroporated, and allowed to recover at 30°C for 3 h before plating onto selective media. Expression of recombinant IFN--y in E. coli. Overnight cultures of E. coli JM109(pGEXIFN) were diluted 1/50 in Luria-Bertani (LB) broth supplemented with 50 ,ug of ampicillin per ml. The cultures were grown at 370C with agitation for 2 h, and then the inducer isopropyl-,3-D-thiogalactopyranoside (IPTG) (Pharmacia) was added to a final concentration of 0.2 mM. After 3 h, the cells were harvested by centrifugation and resuspended in phosphate-buffered saline (PBS). Cells

* Corresponding author. Mailing address: CSIRO, Division of Animal Health, Private Bag No. 1, PO, Parkville, Vic., 3052, Australia. Phone: 03 342 9789. Fax: 03 347 4042. t Present address: AMRAD Corp., 17-27 Cotham Road, Kew, Vic., 3130, Australia.

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were lysed by sonication (1 min, 10 jim, on ice), and cellular debris was removed by centrifugation. Expression of recombinant IFN--y in C. glutamicum. Cultures were grown overnight at 30°C in LB broth with 50 jig of kanamycin per ml or 50 jig of hygromycin per ml. Cultures were inoculated as 1/25 dilutions of overnight cultures into LB broth with antibiotics. After 2 h of incubation at 30°C with aeration, IPTG was added to a final concentration of 5 mM. After a further 4 h of incubation, cells were harvested by centrifugation, resuspended in PBS, and sonicated (3 min, 10 jim, on ice). To maximize the amount of soluble recombinant protein recovered for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting (immunoblotting), cells were boiled for 5 min in PBS-1.0% SDS and centrifuged. Cell lysates used for affinity chromatography were cleared after sonication in PBS-Triton X-100 (0.1%). Control of tac promoter in C. glutamicum. C. glutamicum was cotransformed with pTB294 and pPGIFN. Both plasmids were maintained by antibiotic selection with kanamycin (50 ,ug/ml) for pPGIFN and hygromycin (100 jig/ml) for pTB294. Dual antibiotic selection was maintained in agar plates, seed cultures, and expression cultures. The plasmid pTB294 (8), is a derivative of pEP2 which carries a gene for hygromycin resistance and a version of lacI, designated lacIP8 .lacIl8K differs from lacIq in that the promoter, ribosome binding site, and GTG initiation codon have been replaced with a promoter, ribosome binding site, and ATG from the 18-kDa antigen gene from Mycobacterium leprae (2). Affinity purification of GST-IFN--y. Recombinant GST fusion protein (GST-IFN-y) was recovered from lysates by use of columns of glutathione-Sepharose (Sigma). GST-IFN-y was eluted from the Sepharose column with 5 mM reduced glutathione (Sigma) in 50 mM Tris HCl (pH 9.6) or, alternatively, IFN--y was cleaved from GST by digestion with thrombin (Boehringer Mannheim) and eluted from the column with PBS. Protein analysis. The sizes of the recombinant products were estimated by SDS-PAGE (11) after gels were stained with Coomassie brilliant blue R (Sigma). The percentage of recombinant protein in cell extracts was estimated with a Hoefer GS-300 scanning densitometer to scan stained gels. Estimations of protein concentrations in solutions were made with the bicinchoninic acid protein assay (20). Immunological analysis. E. coli transformants were patched onto nitrocellulose discs laid on LB agar (50 jig of ampicillin per ml). Following overnight incubation at 37°C, the discs were placed on filter paper soaked in 10 mM IPTG and laid on fresh agar plates. After three hours at 37°C, the cells were lysed and blotted (9). Western blotting of proteins from SDS-PAGE gels to nitrocellulose was carried out with a Bio-Rad Mini Transblot electrophoretic transfer cell according to the manufacturer's instructions. Nitrocellulose filters, supporting either lysed colonies or Western-blotted proteins, were blocked with 5% skim milk in PBS (PBSB) overnight at 4°C. E. coli antigens were absorbed from rabbit anti-bovine IFN--y polyclonal serum by dilution of the serum in PBSB and incubation at room temperature with nitrocellulose impregnated with lysed E. coli or C. glutamicum cells. Western blots and colony blots were probed with antiserum, washed, and then incubated with sheep anti-rabbit immunoglobulin G antibody conjugate labelled with horseradish peroxidase (Silenus). After the excess conjugate was washed away, 3,3',5,5'-tetramethylbenzidine substrate was incubated with the blots until the positive control was visible. Reactions were stopped by washing the blots in water. Bovine IFN-y (lot AE 62; Ciba-Geigy Ltd.) was used as a positive

kDa

A

B

45_

D

E

F

U.

-

31

C

-

Am"0}00

FIG.1. Coomassie blue-stained SDS-PAGE showing GST-IFN-y~~~~~~~~~~~~~~~~~~~ 21~ F, thrombin-cleavedi;GST-IFN-y. GST;~ I_

14fl,

-

4

blue-stained SDS-PAGE showing Coomassie FIG. 1.a_ GST-IFN-py as neatv control used~~~~ expressed in E. coli. Lanes: A, molecular mass markers; B, uninduced cell lysate; C, induced cell lysate; D, affinity-purified GST-IFN--y; E, GST; F, thrombin-cleaved GST-IFN--y.

control, and GST produced by E. coli JM109(pGEX-2T) was used as a negative control. IFN-y assays. The activities of affinity-purified proteins were measured in an enzyme immunoassay (EIA) with monoclonal antibodies specific for bovine, ovine, and caprine IFN-,y (17, 23). The biological activity of recombinant ovine IFN-,y from E. coli was assessed in an antiviral bioassay (22). International units of activity represent the amount of IFN--y required to protect 50% of cells from lytic infection in an antiviral assay. In the EIA, units of activity were measured against a bovine IFN-y reference standard (lot AE 62, 2.5 x 105 U/mg; Ciba-Geigy Ltd.). In each assay, at least two wells in a serial 1/2 dilution series were required to give positive results for the sample to be considered active. RESULTS

Cloning and expression in E. coli. Part of the gene for ovine IFN-,y encoding the mature protein was subcloned into pGEX2T, resulting in a clone, pGEXIFN, from which IFN-y could be expressed as an in-frame fusion with GST. Expression was initially detected by immunoblotting induced bacterial colonies on nitrocellulose. The molecular masses of GST-IFN-y (42 kDa) and thrombin-cleaved GST-IFN--y (tIFN--y, 16 kDa) were estimated from Coomassie brilliant blue-stained SDSPAGE gels (Fig. 1) and corresponded with those predicted from the IFN--y amino acid sequences (14, 16). Scanning densitometry of PBS-soluble, cytoplasmic protein extracts showed that GST-IFN--y represented 17% of PBS-soluble cellular proteins. IFN--y, the minor component of the fusion protein, represented 4.7% of the cellular proteins. Affinity purification of lysed bacterial cells yielded 57 mg of pure, soluble GST-IFN-y per liter of culture. IFN-y could be cleaved from the GST moiety by digestion of GST-IFN-y with thrombin. Cloning and expression in C. glutamicum. For cloning ovine IFN-y in C. glutamicum, hybrid plasmids containing pEP2 and pGEXIFN were constructed and tested for GST-IFN-y expression in E. coli. Only clones in which pEP2 was in the same orientation as the GST-IFN--y open reading frame expressed GST-IFN--y. The plasmid from expressing clones, pPGIFN, was introduced into C. glutamicum, and transformants were tested for the ability to express GST-IFN--y by growth in liquid medium and induction with IPTG. Extracts of sonicated cells were electrophoresed on SDS-PAGE, Western blotted, and immunoprobed with rabbit anti-IFN--y serum. Of 32 C. glutamicum transformants tested, only half could be induced to express detectable levels of GST-IFN--y (Fig. 2A). No GST-

EXPRESSION OF IFN IN E. COLI AND C. GLUTAMICUM

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C

TABLE 1. Activities of IFN-y from E. coli and C. glutamicum detected in cell lysates and purified protein Host

A

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Protein

E. coli E. coli E. coli

GST-IFN-y

C. glutamicum

GST-IFN-y

tIFN--y GST

Biological activity (U/mg of protein) Purified Purified antiCell lysate EIA viral assay EIA

1.0 x 105 3.1 X 105 4.8 x 105 NDa 0 0 3.0 x 104 ND

3.2 x 105 2.6 x 106 0 ND

a ND, not done.

B FIG. 2. Western blots of induced cell lysates of 12 C. glutamicum pPGIFN isolates. (A) Unstable expression without control; (B) stable expression in cells with pTB294 coresident with pPGIFN. Lane C, negative control.

IFN-y was detected in uninduced cultures. Expressing clones lost the ability to express GST-IFN--y upon subculture. Plasmid DNA extracted from those clones was transformed into E. coli, and all transformants expressed recombinant GST-IFN--y at the same level as an E. coli clone containing pPGIFN which had not been passaged through C. glutamicum. Additionally, restriction endonuclease digestion of the passaged plasmids showed the same pattern as digestion of plasmids that had not been in C. glutamicum, indicating that there had not been any major structural rearrangements in the plasmids. Use of lacl8K to stabilize GST-IFN--y expression in C. glutamicum. While little is known about promoter function in C. glutamicum, it has been noted that promoters of grampositive bacteria may differ slightly from those of gramnegative bacteria. The vector pGEX-2T has the lacIq version of lacI, whose promoter has enhanced activity in E. coli. We transformed C. glutamicum clones with a plasmid carrying lacIl8K, whose promoter is from the gram-positive bacterium M. leprae, to test if possible enhanced LacI-repressor protein production would increase repression of the tac promoter. The plasmid pTB294, bearing both lacI18K and a gene for hygromycin resistance, was transformed into C. glutamicum. Transformants were then transformed with pPGIFN. Transformants resistant to both antibiotics, hygromycin for pTB294 and kanamycin for pPGIFN, were selected and tested for inducible expression of GST-IFN--y. All 12 clones tested expressed GST-IFN-y and maintained that feature upon subculture (Fig. 2B). Detection of biological activity. Purified recombinant IFN--y, recovered from E. coli, was active in vitro, both in a GST fusion protein and when cleaved from GST. GST-IFN--y purified from C. glutamicum was also active in vitro. Activity of recombinant IFN--y was detected in the IFN--y EIA and in an antiviral bioassay (Table 1). An estimation of the units of IFN--y activity in induced cell lysates of C. glutamicum pPGIFN and E. coli pGEXIFN was made with the EIA. Cleared lysates from sonicated cells were titrated in the EIA with cell lysates from clones carrying the vector only as a negative control and bovine IFN-y to make a standard curve. E. coli pGEXIFN lysates contained 0.1 U of IFN-y per ng of total cellular protein, whereas C. glutamicum pPGIFN lysates contained 0.03 U/ng of cellular protein. Only several micrograms of purified GST-IFN--y could be recovered from 1 liter of induced C. glutamicum culture. Estimates of the specific activities were calculated with bovine IFN--y (2.5 x 105 U/mg) as a reference standard and

positive control. GST was used as a negative control and did not react in the assays. As only small amounts of pure GST-IFN--y could be recovered from C. glutamicum pPGIFN, accurate estimations of specific activity were not made. However, GST-IFN-y recovered from C. glutamicum protected cells from viral infection in the antiviral assay and was recognized in the EIA. DISCUSSION Ovine IFN-y was expressed in a GST fusion protein in two bacterial hosts, E. coli and C. glutamicum. E. coli clones carrying pGEXIFN were induced to express GST-IFN--y at levels up to 17% of the soluble cellular proteins. The GST fusion protein was readily purified by affinity chromatography and could be digested by thrombin to release IFN-,y from the GST carrier protein. C. glutamicum was also able to express large amounts of GST-IFN--y; however, problems were experienced in the purification of GST-IFN--y and in clone stability. While stable expression was achieved by enhancing promoter control, the yield of purified recombinant protein remained low compared with that of E. coli. The monoclonal antibodies used in the EIA have previously been shown to recognize only active forms of bovine IFN-y (23). Both versions of recombinant ovine IFN--y, GST-IFN--y and tIFN--y, were recognized by the monoclonal antibodies in the EIA, suggesting that they were biologically active. This was confirmed in antiviral assays in which GST-IFN--y and tIFN--y showed antiviral activity by protection of MDBK cells from lytic infection with Semliki Forest virus. Cells of bovine origin were used in the assay, and therefore protection by ovine IFN--y further demonstrates the cross-specificity of ovine and bovine IFN-y. Additionally, both GST-IFN--y and tIFN--y proteins were detected in Western blots of SDS-PAGE gels with polyclonal serum raised against bovine IFN--y, further demonstrating the high level of structural conservation. SDS-PAGE of lysates from induced E. coli and C. glutamicum clones showed two new bands compared with uninduced controls. The 42-kDa band corresponded to GST-IFN-y, and the other was approximately 27 kDa. Each protein was recognized by both anti-GST serum and anti-IFN-y serum, suggesting that the smaller band is a degraded form of GST-IFN-,y (Fig. 1 and 3). The expression plasmid pGEXIFN was modified for introduction into C. glutamicum by ligation with a shuttle vector to produce pPGIFN. Unlike expression in E. coli clones, expression of GST-IFN-y was unstable in C. glutamicum. C. glutamicum AS019 is wild type with respect to recombination ability, and it was possible that the plasmids were being rearranged. However, plasmid DNA that was recovered from nonexpressing clones and digested with restriction endonucleases appeared identical to the DNA of the original plasmid. Addition-

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kDa

A

B

97 66 45 31 -

-

21 FIG.

3.

Uninduced

-

(lane A)

and induced

(lane B)

cell

lysates

of

C.

glutamicum pPGIFN.

suitable for making large amounts of recombinant IFN-,y from C. glutamicum, and modification of the expression system will be required to exploit C. glutamicum to its full potential. Clearly, a well-controlled promoter will be essential for such an expression system. Secretion of heterologous proteins from C. glutamicum has been demonstrated (12), although other gram-positive bacteria, particularly Bacillus subtilis, have also been used for secretory expression. Given adequate control of expression and a suitable secretion vector, C. glutamicum should be capable of secreting active cytokines cheaply in large-scale industrial fermentations, free of the endotoxins of gramnegative culture systems. ACKNOWLEDGMENTS

ally, plasmids from nonexpressing clones were able to direct IFN--y expression when reintroduced into E. coli, indicating that there had not been any major rearrangements in the plasmids and that the lack of detectable expression was not a result of plasmid instability or mutations in the plasmid. Mutations in the host are probably responsible for the loss of expression. The promoter of lacI (lacIp) is inefficient in E. coli, and consequently the lac promoter (lacp) is not tightly regulated

(3). The lacIq gene that occurs on pGEX-2T has a mutation in the promoter that increases the efficiency of LacI production and better regulates lac-based promoters (3). There is no detailed information on lac promoters in C. glutamicum, although it has been demonstrated that they function in that bacterium and can be regulated by lacI (21). The instability of GST-IFN--y expression in C. glutamicum may be an indication that the more powerful tac promoter (tacp) is not tightly controlled in this host. If low levels of transcription continue constitutively in uninduced bacteria, then resulting selection pressure may lead to nonexpressing clones. A version of lacI, lacId8, with a promoter from M. keprae was tested for increased repression of the tac promoter in C. glutamicum. All C. glutamicum clones containing the plasmids pPGIFN and pTB294 carrying lacIlNK stably expressed GSTIFN--y. Clones could be subcultured and retained the capacity for inducible expression of GST-IFN-y. Both M. leprae and C. glutamicum are gram-positive bacteria of the actinomycete family, and thus we anticipated that the M. leprae promoter would be more efficient than lacIqp. The proposed mechanism of stabilization of expression in this case may be that more Lacd was synthesized, saturating the operator site and resulting in tighter inhibition of tacp. However, it should be noted that both pPGIFN and pTB294 are based on the same replicon from pEP2. Although they were maintained in the same cell by dual antibiotic selection, the copy number of each plasmid is possibly half that which it would be if there were only one pEP2-based plasmid in residence. If a leaky promoter is the problem, then when pTB294 is coresident with pPGIFN and hence when there are half as many pPGIFN plasmids present, the concentration of deleterious gene products in the cell may be reduced to a tolerable level and the pressure on the population to select for nonexpressers would be reduced. GST-IFN--y recovered by affinity purification of C. glutamicum lysates was biologically active, as shown in the EIA and the antiviral assay. Recovery of pure GST-IFN-y from C. glutamicum was not as high as that from E. coli. After sonication and centrifugation, a large proportion of the recombinant protein remained in the pellet. This suggests that it accumulated as inclusion bodies in C. glutamicum which were not recovered. The pGEX system in its present form is not

H. Billman-Jacobe was supported by a Junior Research Fellowship from the Meat Research Corporation of Australia. We thank Jim Rothel and Lyn Hurst for assistance with EIAs and bioassays.

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