construction of fusion vectors of corynebacteria: expression of glutathione-s-transferase fusion...

Construction of fusion vectors of corynebacteria:expression of glutathione-S-transferase fusion protein in

Corynebacterium acetoacidophilum ATCC 21476

Preeti Srivastava, J.K. Deb �

Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology, Delhi, New Delhi 110 016, India

Received 25 October 2001; received in revised form 29 April 2002; accepted 4 May 2002

First published online 29 May 2002

Abstract

A series of fusion vectors containing glutathione-S-transferase (GST) were constructed by inserting GST fusion cassette of Escherichiacoli vectors pGEX4T-1, -2 and -3 in corynebacterial vector pBK2. Efficient expression of GST driven by inducible tac promoter of E. coliwas observed in Corynebacterium acetoacidophilum. Fusion of enhanced green fluorescent protein (EGFP) and streptokinase genes in thisvector resulted in the synthesis of both the fusion proteins. The ability of this recombinant organism to produce several-fold more of theproduct in the extracellular medium than in the intracellular space would make this system quite attractive as far as the downstreamprocessing of the product is concerned. 9 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. Allrights reserved.

Keywords: Corynebacterium; Glutathione-S-transferase fusion vector; Enhanced green £uorescent protein; Streptokinase

1. Introduction

Escherichia coli has been the workhorse of genetic en-gineers both for basic studies as well as for the expressionof recombinant proteins. There are some limitations ofE. coli as a recombinant host. Normally, the organismdoes not secrete protein. This is not advantageous fromthe downstream processing point of view. Also, it is not afood grade bacterium. In this respect, Bacillus subtiliscould be useful but it has high proteolytic activity. Alter-natively, Bacillus brevis has been used for this purpose [1].Secretory nature and no detectable extracellular proteaseshave been an advantage of this organism. Generally, foodgrade organisms such as Lactococcus lactis [2,3] are usefulfor the production of commercially important proteins.

Soil corynebacteria, such as Corynebacterium glutami-cum, are another class of food grade bacteria that canserve as potential hosts for the production of recombinantproteins. During the past two decades, considerable prog-ress has taken place in our understanding of the molecular

biology of coryneform bacteria through the developmentof e¡ective transformation systems and cloning vectors.Most of these vectors were constructed from endogenousplasmids [4,5]. These plasmids varied over a wide molec-ular range, from as low as 3 kb to as high as 55 kb.Exhaustive reviews on the molecular biology of these plas-mids and cloning vectors constructed with them have ap-peared in recent years [6,7]. Considerable progress hasbeen made in the development of C. glutamicum^E. colishuttle vectors, corynebacterial expression vectors forcloning of amino acid biosynthesis pathway genes andpromoter probe vectors [8,9]. Also, expression of heterol-ogous proteins such as B. subtilis protease subtilisin hasbeen achieved in corynebacteria using vector pEP2 [10].The gene encoding ¢bronectin binding protein 85A of My-cobacterium tuberculosis has also been expressed in C. glu-tamicum using pBL1 replicon based vectors containing tacpromoter of E. coli or endogenous cspB gene promoter[11]. It was reported earlier by Morinaga et al. [12] thatcommon E. coli promoters such as lacUV5, tac and trpfunction very well in Brevibacterium lactofermentum. Theformer two promoters have been found to be 50% activein this organism compared to their activity in E. coli.Later, search for endogenous promoters has led to thecloning and identi¢cation of promoters in C. glutamicum

0378-1097 / 02 / $22.00 9 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.PII: S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 7 4 0 - 1

* Corresponding author. Tel. : +91 (11) 6591006;Fax: +91 (11) 6868521.

E-mail address: [email protected] (J.K. Deb).

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FEMS Microbiology Letters 212 (2002) 209^216

www.fems-microbiology.org

[13]. The study also revealed normal consensus sequencesat about 35 bp and 10 bp upstream of the transcriptionstart site, similar to that observed in many other bacterialgenes. These promoters were also found to be functionalin E. coli.

Earlier reports from this laboratory described the con-struction of several vectors, including corynebacteria^E. coli shuttle vector, based on pBL1 replicon [14,15]. Adetailed study on the stability of these vectors in Coryne-bacterium acetoacidophilum in continuous culture showedthe vectors to be extremely stable. The study also led tothe identi¢cation of minimum replicon. Since none ofthem were expression vectors, we were interested in ex-ploiting similarities in promoter functions in E. coli andin corynebacteria for the construction of diverse expres-sion vectors using strong tac promoter of E. coli. One ofthe useful strategies for the expression of heterologousprotein is to construct a fusion vector that would resultin the expression of foreign gene as a fusion product. Thisis favored under such circumstances where the expressionof heterologous protein in the host is di⁄cult for variousreasons. The fusion protein produced can sometimes bepuri¢ed by a⁄nity chromatography. One of the genes ex-tensively used for this purpose in E. coli is glutathione-S-transferase (GST). To explore if the construction offusion vector based on GST would lead to successful ex-pression of this enzyme in corynebacteria, we constructedvectors using tac promoter and GST gene in C. acetoaci-dophilum. Our ultimate objective was to clone, express andpurify heterologous protein in corynebacteria. The presentwork describes the construction of GST fusion vectors,study of expression of GST in C. acetoacidophilum andof genes for green £uorescent protein and streptokinaseas GST fusion proteins.

2. Materials and methods

2.1. Bacterial strains, plasmids and growth conditions

C. acetoacidophilum ATCC 21476 was the corynebacte-rial strain used in the present study. E. coli DH5K wasused as the host for transformation by E. coli plasmids.E. coli plasmids used in the present study were pTrc99A,pEGFPC1, pUCsk and pGEX4T-1^3. For the construc-tion of fusion vectors, the corynebacterial plasmid pBK2containing pBL1 replicon was used. Both C. acetoacido-philum and E. coli were maintained in Luria agar (LA) andwere routinely grown in Luria broth (LB). The transform-ants were grown in the medium supplemented with appro-priate antibiotics at the following concentrations: ampicil-lin (40 Wg ml31) and kanamycin (40 Wg ml31). Brain-heartinfusion (BHI) and hypertonic brain-heart infusion media[14] were used respectively for the growth and preparationof protoplasts of C. acetoacidophilum.

2.2. Recombinant DNA techniques

All DNA manipulations were carried out according toSambrook et al. [16]. Restriction enzymes were obtainedfrom Boehringer Mannheim, Germany and MBI Fermen-tas. Restriction digestion was carried out according totheir protocols. Puri¢cation of DNA from agarose gelswas carried out using Qiaquick gel extraction kit (Qiagen,Chatsworth, GA, USA). For blunt end ligation, the vectorDNA was treated with calf intestinal phosphatase (CIP)and the dephosphorylated DNA fragments were puri¢edby Qiaquick column (Qiagen).

2.3. Transformation of E. coli and C. acetoacidophilum

Transformation of CaCl2 treated E. coli cells was car-ried out according to Sambrook et al. [16]. Preparationof protoplasts of C. acetoacidophilum and their transfor-mation were carried out according to Mukherjee et al.[15].

2.4. Plasmid stability studies

Single colony of plasmid bearing cells was inoculated inBHI medium containing appropriate antibiotic and al-lowed to grow overnight at 30‡C. The overnight grownculture was inoculated in 100 ml of fresh BHI medium(0.5% inoculum) and was grown without selection pressurefor another 24 h. This culture was used as inoculum forfresh 100 ml BHI medium and the above process repeatedfor 3 days. Every day, a sample of overnight grown culturewas serially diluted and aliquots of appropriately dilutedsamples were plated on BHI agar plates. The coloniesfrom these plates were replica plated on BHI agar plateswith and without antibiotics. The fraction of plasmidbearing cells was calculated by dividing the number ofcolonies growing on antibiotic-containing plates by thosegrowing on antibiotic-free plates.

2.5. Preparation of cell extracts

E. coli cells harboring pGEX plasmid were grown in LBcontaining ampicillin (40 Wg ml31) at 37‡C. C. acetoacido-philum harboring plasmids were grown in BHI mediumcontaining kanamycin (40 Wg ml31) at 30‡C. The exponen-tially growing cells were subjected to IPTG (1 mM) induc-tion and the cells were harvested after desired time bycentrifugation and washed several times with 20 mM po-tassium phosphate bu¡er (pH 7.0). E. coli cells were bro-ken by 30 s each of alternate sonication and chilling for atotal duration of 10 min. The same procedure was fol-lowed for corynebacteria except that the duration in thiscase was 30 min. The sonicated cell suspension was cen-trifuged at 10 000 rpm for 15 min and the supernatantused for GST assay.

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P. Srivastava, J.K. Deb / FEMS Microbiology Letters 212 (2002) 209^216210

2.6. Assay of GST activity

The activity of the enzyme in the cell extract was deter-mined by the method utilizing glutathione and 1-chloro-2,4-dinitrobenzene (CDNB) as substrates for GST. Theyellow product S-(2,4-dinitrophenyl)glutathione formedwas detected by measurement of absorbance at 340 nm[17].

2.7. Screening of clones for streptokinase

The transformants were transferred from hypertonicBHI plates to LA plates and incubated overnight at30‡C. The plates were overlaid with molten agar contain-ing defatted milk and plasminogen, incubated at 30‡C andpositive clones were identi¢ed by the zone of clearancearound the colonies.

Fig. 1. Construction of corynebacterial GST fusion vector pBKGEXm2. Gray bar, the larger fragment of pBK2; black bar, the smaller fragment ofpGEXm2.

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P. Srivastava, J.K. Deb / FEMS Microbiology Letters 212 (2002) 209^216 211

2.8. Streptokinase assay

Streptokinase activity was determined by a method sim-ilar to that described by Castellino et al. [18]. ChromozymPL (Boehringer Mannheim) was used as the substrate.

Brie£y, the cell extract containing streptokinase was mixedwith plasminogen and pre-incubated at 37‡C for 5 min.The substrate was then added, and the release of 4-nitro-aniline was monitored by measuring absorbance at 405nm.

Table 1Intracellular and extracellular activities of GST

Sample Intracellular GST activity Extracellular GST activity Total activity (U) Extracellular/intracellular

U ml31 Total U U ml31 Total U

E. coli (DH5K) pGEX4T-2 100 100 2.0 41.6 141.6 0.4C. acetoacidophilum pBKGEXm2 89.5 89.5 20.8 416.6 506.1 4.6

Fig. 2. Construction of corynebacterial GST^GFP fusion vector pBKGEXm2EGFP.

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2.9. Sodium dodecyl sulfate^polyacrylamide gelelectrophoresis (SDS^PAGE)

Proteins were fractionated by electrophoresis in a 12%SDS^polyacrylamide gel according to the method ofLaemmli [19].

3. Results and discussion

3.1. Construction of GST fusion vectors of corynebacteria

For the construction of a GST fusion vector, the plas-mid pGEX4T-2 was initially modi¢ed to generatepGEXm2. For this purpose, pGEX4T-2 was digested bySspI and the smaller fragment containing the GST cassettewas puri¢ed by gel elution. Similarly, pTrc99A was di-gested with SspI to generate two fragments and the largerfragment carrying the lacIq gene, the pBR322 origin ofreplication and the ampr gene was gel eluted and dephos-phorylated by CIP. The two gel eluted fragments wereligated to obtain the plasmid pGEXm2. This plasmidhas a single SphI site which is derived from the pTrc99Afragment. The above manipulation made it possible to getthe lacIq regulator gene and the GST cassette in a singlefragment when pGEXm2 was digested with SphI andScaI. Since pBK2 has multiple closely spaced SphI sitesin the non-essential region of the plasmid, the above frag-ment will have a compatible cohesive end for insertion inpBK2. Initially, pBK2 was digested with MluI and bluntended with Klenow DNA polymerase. Subsequent diges-tion by SphI generated two fragments. The larger frag-ment containing the pBL1 origin of replication was ligatedto the SphI^ScaI fragment containing the lacIq and GSTcassette of pGEXm2 to obtain pBKGEXm2 (Fig. 1). Thesame strategy was followed to construct pBKGEXm1 andpBKGEXm3 ¢rst by constructing pGEXm1 and pGEXm3respectively.

3.2. Expression of GST

To check if Ptac-driven GST can be expressed inC. acetoacidophilum, recombinants were obtained by trans-formation of the protoplasts with pBKGEXm2 and isola-tion of kanamycin-resistant colonies. Stability studiesshowed that the plasmid was 100% stable up to 60 gener-ations (data not shown). For extraction of enzymes fromcells, the recombinant cells were grown in hypertonic BHImedium in the presence of 40 Wg ml31 kanamycin. Whenthe cells reached an OD600nm of 0.4, induction by IPTGwas initiated. The cells were harvested at di¡erent timesafter the induction and enzyme assays carried out on thecell-free extracts as described in Section 2. The optimumtime for harvesting of cells after induction was found to be3 h. Cell extract was also prepared from E. coli DH5Kbearing pGEX4T-2 of approximately the same cell density

as that of C. acetoacidophilum cells. This served as positivecontrol. Extracts of E. coli DH5K and C. acetoacidophilumharboring pBK2 were used as negative controls. Althoughthe intracellular GST activities in the two strains werecomparable, the total GST activity of C. acetoacidophilumrecombinant was 3.5 times more than that of E. coli (Ta-ble 1) in spite of the fact that the copy number of E. coliplasmid pGEX4T-2 was higher than that of C. acetoaci-dophilum plasmid pBKGEXm2 (data not shown). This isquite remarkable, since it was earlier found that tac pro-moter-driven expression of CAT activities was the same inboth B. lactofermentum and E. coli [12]. Moreover, it wasobserved that though the intracellular GST activity wascomparable in the two strains, the extracellular activitiesdi¡ered considerably. The bulk of the activity was intra-cellular in E. coli, but the intracellular activity in C. ace-toacidophilum was 20% of its extracellular activity. Theseresults show that the enzyme is mainly secreted by thelatter strain, which is quite attractive from the downstreamprocessing point of view. It was also observed that some-times heterologous genes need modi¢cation of their co-dons to suit those of the host for optimum expression[20]. Our results suggest that the native codons of GSTare recognized e⁄ciently by C. acetoacidophilum. The bet-ter expression of GST in C. acetoacidophilum may be dueto more e⁄cient transcription from Ptac or due to e⁄cienttranslation of GST codons or both.

3.3. Expression of EGFP fusion protein inC. acetoacidophilum

Since Ptac-driven GST is e⁄ciently expressed in C. ace-toacidophilum, it would also be of interest to study theexpression of heterologous protein as fusion partner ofGST in this host. The green £uorescent protein EGFP

Fig. 3. SDS^PAGE of induction of EGFP by recombinant C. acetoaci-dophilum : Lane 1, uninduced; lane 2, induced; lane M, molecular massmarkers. GST^EGFP fusion protein band is indicated by an arrow.

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was chosen for this purpose due to its diverse applications.The fusion vector was constructed by digesting plasmidpEGFPC1 sequentially with MluI and NheI. The smallerfragment was gel eluted and the ends were ¢lled with Kle-now DNA polymerase. This fragment was inserted at theSmaI site of pBKGEXm2. This would allow 50% of thepopulation of the insert to be joined in the right orienta-tion and in the right frame. The resulting vectorpBKGEXm2EGFP (Fig. 2) was used to transform C. ace-toacidophilum and the transformants screened by £uores-

cence at an excitation wavelength of 480 nm. The positiveclones expressing a functional GFP were identi¢ed bygreen £uorescence. The fusion protein appeared as an in-tense band in polyacrylamide gel of the IPTG-inducedsample (Fig. 3). There are several versions of GFP avail-able. Genes like GFPuv contain codons which are pre-ferred in E. coli, while others such as EGFP, used in thepresent study, contain silent base mutations that corre-spond to human codon usage preferences. Leblon and co-workers [21] reported expression of GFPuv in C. glutami-

Fig. 4. Construction of corynebacterial GST^streptokinase fusion vector pBKGEXm2sk. Striped bar, the HincII fragment containing streptokinase gene(sk).

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cum. Sun et al. [22] reported that EGFP is expressed e⁄-ciently in Streptomyces coelicolor due to the codon usageof jelly¢sh, the natural source of this protein, correspondsto that of many GC-rich Streptomyces genes. The presentwork showed that such codons are also expressed well incorynebacteria although the GC content of C. acetoacido-philum is not as high as that of S. coelicolor. The resultsare important because they indicate that it is possible toexpress heterologous eukaryotic genes e⁄ciently in cory-nebacteria as far as this protein is concerned.

3.4. Expression of streptokinase fusion protein inC. acetoacidophilum

Streptokinase is a thrombolytic drug extensively used ase¡ective therapy for improving survival and preserving leftventricular function [23]. High-level expression of strepto-kinase has been achieved in various laboratories usingE. coli as host [24]. At the industrial level also recombi-nant E. coli is used to produce streptokinase. Also, thereare several reports of recombinant streptokinase producedin B. subtilis [25]. It was demonstrated by Klessen et al.[26] that the HincII fragment of streptokinase is function-ally active. Since GST was mostly secreted into the me-dium by C. acetoacidophilum, it would be of interest toexpress streptokinase as a GST fusion product to checkif the fusion protein is also e⁄ciently secreted. We usedthe HincII fragment of the streptokinase gene for the con-struction of streptokinase fusion vector. The fragment wasretrieved from streptokinase gene cloned in an E. coli plas-mid pUCsk, which contains a 2.5 kb PstI fragment bear-ing the streptokinase gene cloned at the single PstI site ofpUC19. The plasmid was digested with BanI which formsseveral small fragments and one large fragment. The latterwas puri¢ed, digested with HincII and the fragment carry-ing the streptokinase gene was cloned at the SmaI site ofplasmid pBKGEXm2. The resulting vector pBKGEXm2sk(Fig. 4) was introduced into C. acetoacidophilum cells byprotoplast transformation and clones were selected for ka-namycin resistance. The positive clones were identi¢ed bythe assay based on clear zones formed around the colonieson plasminogen^milk agar plates. The determination ofstreptokinase activities in the intracellular extract of thecells and in the culture medium showed that more than90% of the activity was secreted out into the medium(Table 2). The ratio of extracellular to intracellular strep-tokinase activities was higher than that observed withGST. The GST^streptokinase fusion protein thus synthe-sized is amenable to puri¢cation by a⁄nity chromatogra-

phy on glutathione^Sepharose column. The advantage ofthe system thus developed is that the presence of the bulkof the activity in the medium would make the downstreamprocessing of this protein cheaper since the recovery ofprotein by an energy-intensive cell disruption process canbe avoided.

Acknowledgements

This study was supported by a ¢nancial grant (No. SP/SO/D-68/96) from the Department of Science and Tech-nology, Government of India to J.K.D.

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Table 2Intracellular and extracellular activities of streptokinase

Sample Intracellular Extracellular Total activity (U) Extracellular/intracellular

U ml31 Total U U ml31 Total U

C. acetoacidophilum pBKGEXm2sk 36 1800 560 28 000 29 800 15.6

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construction of fusion vectors of corynebacteria: expression of glutathione-s-transferase fusion...

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