Improved synthesis of Salmonella typhimurium enterotoxin using gene fusion expression systems

Gene, 144 (1994) 81-85 0 1994 Elsevier Science B.V. All rights reserved.

81

0378-1119/94/$07.00

GENE 07898

Improved synthesis of Salmonella typhimurium enterotoxin using gene fusion expression systems (Virulence factor; glutathione S-transferase; anti-peptide antibodies; phage T7 RNA polymerase/promoter thioredoxin; DNA sequencing; DNA supercoiling)

system; PCR;

A.K. Chopra”, A.R. Brasierb, M. Dasa, Xin-Jing Xu” and J.W. Petersona aDepartment of Microbiology and Immunology and bSealy Center for Molecular Science, Internal Medicine and Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, TX 77555-1019, USA Received by A.M. Chakrabarty:

25 October

1993; Revised/Accepted:

3 January/l8

January

1994; Received at publishers:

21 February

1994

SUMMARY

Salmonella

enterotoxin (Stn) is a virulence factor in S. typhimurium strain Ql that causes both fluid secretion in ligated intestinal loops of rabbits and elongation of Chinese hamster ovary (CHO) cells. High-level expression systems are needed to provide Stn in soluble form for detailed study of the biological activity of Stn. To maximize the synthesis and solubility of Stn, we systematically compared the production of native Stn synthesized with a T7 RNA polymerase/promoter system to that of two fusion proteins: glutathione S-transferase::Stn (Gst::Stn) and thioredoxin A::Stn (TrxA::Stn). The latter fusion protein expression systems resulted in a 64-fold increase in Gst::Stn and TrxA::Stn antigen concentration, as measured by specific anti-peptide antibodies in an enzyme-linked immunosorbent assay (ELISA). Most of the toxin derived using these vector systems was insoluble; however, the solubility of the TrxA::Stn antigen increased by at least 50-fold, with a concomitant increase in CHO cell elongation activity. In addition, stn gene expression was enhanced more than 50-fold by addition of 0.2-0.4 M NaCl to Luria-Bertani medium. The biological activity of Stn also was increased in the high-osmolarity medium. Consequently, the expression of stn may be regulated by DNA supercoiling.

INTRODUCTION

The overall biochemical and genetic properties of typhimurium, which generally causes a self-

Salmonella

Correspondence to: Dr. A.K. Chopra, Immunology 77555-1019, Abbreviations:

of Microbiology

and

University of Texas Medical Branch, Galveston, USA. Tel. (l-409) 772-4987; Fax (l-409) 772-5065.

TX

A, absorbance

Department

(1 cm); aa, amino

acid(s); Ab, antibody(-

ies); Ap, ampicillin; bp, base pair(s); CHO, Chinese hamster ovary; ELISA, enzyme-linked immunosorbent assay; Gst, glutathione S-transferase; gst, C&t-encoding gene; IPTG, isopropyl-B-ri-thiogalactopyranoside; kb, kilobase or 1000 bp; LB, Luria-Bertani (medium); nt, nucleotide(s); PAGE, polyacrylamide-gel polymerase chain reaction; re-, recombinant;

electrophoresis; PCR, SD, Shine-Dalgarno

(sequence); SDS, sodium dodecyl sulfate; Stn, Salmonella enterotoxin; stn, Stn-encoding gene; TrxA, thioredoxin; trxA, TrxA-encoding gene; wt, wild type; [I, denotes plasmid-carrier state; ::, novel junction (fusion or insertion). SSDI 0378-1119(94)00109-6

limiting form of gastroenteritis (Popoff, 1991), have been well characterized. However, the molecular virulence factors, which result in pathological changes in the intestinal mucosa, have not been rigorously established. The production of an enterotoxin by isolates of Salmonella has been reported by several investigators (Groisman et al., 1990). Recently, we have cloned, sequenced and expressed one such enterotoxin-encoding gene (stn) from S. typhimurium strain Ql (Prasad et al., 1990; 1992; Chopra et al., 1994). A gene product Stn* (truncated toxin), expressed with a T7 RNA polymerase/promoter system in Escherichia coli, was missing 38 amino acid (aa) residues at the C terminus. Although Stn was truncated, cell lysates from the E. coli recombinant (re) clones exhibited typical enterotoxic activity, that is, fluid secretion in the rabbit intestinal loop model (Chary et al., 1993). Expression of the stn* gene was increased by creating a

82 typical start codon (ATG) and by modifying the ShineDalgarno (SD) sequence by site-directed mutagenesis (Chopra et al., 1994). However, most of the Stn* in these constructs was insoluble. To circumvent the solubility problem and to facilitate purification of Stn, we hyperexpressed both the complete (stn) and incomplete (stn*) genes by placing them downstream from the gst or trxA genes. The present studies were designed to evaluate the expression of the stn gene using gene fusion expression systems and to examine the solubility of the fusion proteins. Availability of these hyperexpression vectors could facilitate purification of Stn, which is essential for studying the structure and function of this enterotoxin.

1

23456

+ 30 kDa + 25

EXPERIMENTAL

AND DISCUSSION

(a) Comparison of the expression of incomplete (stn”) and full-length stn genes using a T7 RNA polymerase/promotersystem

Fig. 1 depicts the expression of the 35S-labeled polypeptides encoded by the full-length stn (lanes 2-4) and truncated stn* genes (lanes 5 and 6). Based on the nt sequence, the sizes of the Stn* and Stn proteins should be 24256 and 29073 Da, respectively. The deduced size of Stn closely matched that of the observed molecular weight of Stn, based on SDS-PAGE (Fig. 1). These protein bands reacted with anti-peptide Ab made to Stn (Chopra et al., 1994). The minor radioactive bands depict either truncated or degraded products of the stn gene (Prasad et al., 1992; Chary et al., 1993). To quantitate the amount of Stn antigen, an ELISA was performed with ceil lysates of the E. coli clones harboring the incomplete and complete stn genes. No remarkable change was noted in the amount of Stn antigen formed by the E. coli clones synthesizing either the Stn or Stn*. In most experiments, the yield of Stn antigen was somewhat lower in the E. coli clones containing the stn gene compared to those with stn*. In addition, only a small portion of Stn (less than 10%) was in soluble form and the remainder of the toxin protein was pelleted with the cell membranes (Fig. 2). In wt Salmonella, the Stn concentration was low compared to that produced with the T7 expression system. These data suggested that stn gene expression in wt Salmonella was under the control of a weak promoter (data not shown). (b) Hyperproduction vector pGEX-KG

of Stn using gene fusion expression

To maximize expression of the stn gene, we constructed re-plasmids, in which truncated stn* and complete stn genes were placed downstream and in-frame with gst. The

Fig. 1. SDS-PAGE HBlOl [pGPl-21 re-plasmids PstI-EcoRI derivative

of the 3sS-labeled polypeptides cells (Tabor and Richardson,

encoded 1985)

by E. coli harboring

pPE49, pHP5, and pPE6. Plasmid pPE6 contained a LO-kb fragment encoding the stn* gene. Plasmid pPE49 is a of pPE6 with a typical

ATG start codon

and a purine-rich

(AGGAGGA) SD sequence for the stn* gene (Chopra et al., 1994). The pHP5 plasmid contained a 749-bp HindIII-PstI PCR fragment from the original 2.8-kb C&-WI fragment that encoded the complete stn gene (Chopra et al., 1994). The stn re-plasmids pPE6, pPE49, and pHP5 were derivatives

of expression

coli whole cell lysates

pT7-6

and pGPl-2;

vector

pT7-6.

Lanes

l-6

contained

E.

with the following plasmids: 1, control plasmids 2-4, plasmid pHP5 containing the complete stn

gene; 5-6, plasmids pPE49 and pPE6, respectively, containing the stn* gene. The samples were subjected to 0.01% SDS-12% PAGE (Laemmli, 1970), enhanced with Enlightning (DuPont), dried and subjected to autoradiography. Arrows indicate the sizes of the complete (29 kDa) and truncated (25 kDa) Stn protein. The 12-kDa polypeptide represents a truncated

or degradation

product

of Stn (Chary

et al., 1993).

amount of Stn antigen, measured by ELISA, increased 64-fold when synthesized as a fusion protein (Gst::Stn), compared to the amounts of Stn produced with a T7 RNA polymerase/promoter system (Fig. 2). It is conceivable that the epitopes, to which the anti-peptide Ab react, were more accessible in the chimeric proteins than in the

83 I-

I

Dilution reciprocal of Stn Fig. 2. Expression circles, triangles with the plasmids stn”), respectively, broken

of the stn gene using different vector systems. Closed and squares

represent

lines represent

soluble

Stn

pGEX-KG

Stn in E. coli clones

Stn from the above

clones. Plasmid pPE49 is described An ELISA was performed (Prasad insoluble

insoluble

pPE49 (O), pTRXFUS (A) and pGEX (m) (with whereas the corresponding open symbols with

antigen

using

is a derivative

mentioned

E. co/i

in the legend for Fig. 1. Methods: et al., 1992) with the soluble and

fourfold

dilutions.

of plasmid pGEX-2T

Expression

vector

(Guan and Dixon, 1991)

and contains the gst gene which is under the control of a tat promoter. For expression of the stn gene in the pGEX-KG vector system, the ApR E. coli XLl-Blue shaking,

re-clones

were grown

at 37°C in LB medium,

to an A6,,0 nm of 0.2. The culture

was then induced

with

with 1 mM

IPTG for 4 h before harvesting. The E. cob strain G1724, containing the trx.4 fusion expression plasmid (pTRXFUS), was grown at 30°C in M-9 induction medium with 100 pg of Ap/ml ( LaVallie et al., 1993). At an A 600nm of 0.2, the culture was induced for 4 h by adding 100 pg Ltryptophan/ml,

and the temperature

of the culture

To prepare cell lysates from various were sonicated. After centrifugation supernatant antigen.

(with 3 M guanidine)

The bacterial

as the source

of insoluble

was shifted to 37°C.

E. coli re-clones, the bacterial cells at 10 000 x g for 10 min, the clear

was used as the source of soluble Stn

pellet was solubilized Stn. To express

in 3 M guanidine

and used

the stn gene in a T7 RNA

polymerase/promoter system, we followed the method of Tabor and Richardson (1985). All the re-clones grew to an A m0nm-- 10 after 4 h induction. In E. co/i clones, in which the stn gene was under the control of a T7 RNA polymerase/promoter an A 6oonm = 1.0 before induction

system, the culture at 42°C.

efficiency. Further, expression of the gst::stn gene might have been improved under the control of the tat promoter. Most of the Gst::Stn antigen was insoluble and associated with the cell membrane fraction (Figs. 2 and 5). SDS-PAGE analysis of whole cell lysates from E. coli clones, containing the Gst::Stn fusion protein, failed to show a major protein band after Coomassie blue staining of the gel (Fig. 3A, lanes 2,3), suggesting that the gst::stn gene was not highly expressed. However, immunoblot analysis clearly demonstrated a 51- and a 55-kDa fusion protein (Fig. 3B, lanes 2, 3). Other bands in the immunoblot were most likely truncated or degradation products of the fusion protein. Hyperproduction of Gst was clearly demonstrated by Coomassie blue staining of the gel (Fig. 3A, lane 1). These data suggested that Stn might have intrinsic protease activity which resulted in self degradation. Alternatively, Andersen-Beckh et al. (1989) similarly reported that premature termination of translation reduced yields of tetanus toxin in E. coli.

was grown

to

native Stn, resulting in higher ELISA values. To minimize this possibility, we opted to denature (unfold) both the native and chimeric proteins with guanidine and/or SDS before performing ELISAs. We also measured the biological activity of both native and fusion proteins, but no difference was noted (Fig. 5). Further, there was no significant difference in the Stn ELISA values after thrombin cleavage (Guan and Dixion, 1991) of Gst::Stn, indicating that Gst did not interfere with quantitation of the Stn antigen. Interestingly, the amount of Stn* synthesized by E. coZi[pPE49] was less than that of the clone expressing the stn gene fused with gst. The presence of a 345bp noncoding sequence upstream from the start codon of the stn* gene could have resulted in reduced transcription

(c) Hyperproduction of Stn using gene fusion expression vector pTRXFUS To obtain soluble Stn, we constructed another re-plasmid in which the stn gene was placed downstream and in-frame with the trxA gene, which is under the control of a p,_ promoter. In this newly described vector system (pTRXFUS), many hyperproduced proteins have been shown to remain soluble (LaVallie et al., 1993). Fig. 2 demonstrates that the amount of soluble Stn antigen increased substantially (at least 50-fold) after fusion with TrxA; however, a significant proportion of the Stn antigen produced by vectors pGEX-KG and pTRXFUS was insoluble. The biological activity (CHO cell elongation titer) of TrxA::Stn was eightfold more than that of native Stn and Gst::Stn (Fig. 5), further suggesting that the soluble Stn antigen was biologically active. Although there was no visible Coomassie-blue-stained fusion protein band (Fig. 3A, lanes 4, 5), immunoblot analysis demonstrated the presence of Stn* and Stn fusion protein bands having the correct sizes of 37 and 42 kDa, respectively (Fig. 3B, lanes 4,5). (d) Effect of NaCl on the expression of the stn gene To determine the effect of NaCl on the synthesis of Stn, E. cob clones, containing the stn gene, were grown in LB medium supplemented with 0.0-0.6 M NaCl. The amount of Stn* antigen increased more than 50-fold when 0.4 M NaCl was added to the medium (Fig. 4). In contrast, the complete stn gene was expressed maximally (16-fold) at 0.2 M NaCl, but expression decreased at higher salt concentrations. These data suggested that regulation of stn transcription was analogous to that of the inv gene, required for the invasion of Salmonella into epi-

84

1234567 -

-,

T _.

-

123456

2.0

n

0.0 M NaCl

k%Ed 0.2 M NaCl 0.4 M NaCl 0.5 M NaCl 1.5

? z

1.0

5

0.5

0.0

1

4

16

64

256

1024

Dilution reciprocal of Stn (X103)

Fig. 4. Effect of NaCl on Stn synthesis.

E. coli[pGEXstn*]

scribed in the legend to Fig. 2, was induced 0.17 M NaCl)

e Fig. 3. Coomassie SDS-12%

PAGE

blue staining

and immunoblot

of the Gst::Stn

and TrxA::Stn.

analysis

of 0.01%

(A) Coomassie

with gst gene alone; 2, pGEXstn*;

3, pGEXstn;

4,

pTRXFUSstn*; 5, pTRXFUSstn; 6, vector pTRXFUS with trxA gene alone. The sizes of Gst and TrxA are indicated by arrows in lanes 1 and 6. Lane 7 contained the molecular mass markers and the dashes represent the sizes (in Da) of protein bands from top to bottom (200, 97, 69, 46, 30, 21.5 and

14.3). (B) Immunoblot

analysis

clone, de(containing

with O-O.6 M NaCI. After harvesting,

the

bacterial cells were resuspended in SDS-PAGE buffer, boiled and subjected to ELISA using mixed Ab to synthetic peptides B112 and Bll3. Average

ELISA values with standard

errors

from 7-S experiments

are

reported.

blue-

stained gel. Lanes l-6 contained E. coli whole-cell lysates with the plasmids carrying the incomplete and complete stn genes, respectively: 1, vector pGEX-KG

supplemented

in LB medium

of the same

Therefore, it is tempting to speculate that these genes could be coordinately regulated by salt concentration in the medium and/or intestinal environment. Although expression of the stn gene could be increased using a gst-gene fusion expression system, the biological activity of the toxin was largely unaltered, due to the insoluble

nature

of the toxin (Fig. 5). However,

at least

samples as in A. The positions of Stn* (lanes 2 and 4) and Stn (lanes 3 and 5) are indicated by arrows. Mixed affinity-purified anti-peptide antibodies

to Stn were used in the immunoblot

1994). Methods:

Polyclonal

residues 47-61) and B112 (aa 196-210), C-termini of Stn, were used in this study biological

activity

analysis

Ab to two synthetic

of Stn was measured

(Chopra

peptides,

et al.,

B113 (aa

representing the N- and (Chopra et al., 1994). The by the CHO

cell elongation

assay (Prasad et al., 1990). The Ab B112 (468pg/ml) and (559 pg/ml) were diluted 1:400 for immunoblots. The membranes

B113 were

developed with an alkaline phosphatase substrate conjugate kit (BioRad, Hercules, CA, USA). The stn gene was amplified by PCR before subcloning into the above mentioned expression vectors. Synthetic oligodeoxyribonucleotides (44-mer), with vector compatible ends, were synthesized, and primers placed downstream and Geneamp described

were designed in-frame with

such that the stn gene was the gene fusion partner. A

reagent kit with AmpliTaq DNA polymerase was used as by the manufacturer (Perkin Elmer Cetus, Norwalk, CT,

USA), and the stn gene sequence was amplified from 40 ng of purified plasmid DNA. After subcloning the PCR product into a suitable vector, the PCR product was nt sequenced by the method of Sanger et al. (1977), using a Sequenase version 2.0 kit (US Biochemical, Cleveland, OH, USA).

thelial cells. Expression of the inv gene was shown to be affected by DNA supercoiling (Galan and Curtiss III, 1990). Since small amounts of Salmonella enterotoxin were synthesized, bacterial invasion of epithelial cells might be crucial for the toxin’s enterotoxic effect.

Fig. 5. Synthesis of Stn using various vector systems. Cell lysates of E. co/i clones with different re-plasmids were prepared as described in legends for Fig. 2. Samples of the cell lysates were added to the CHO cell media (Prasad et al., 1990), and the morphology of the CHO cells was examined after 36 h. The control consisted of cell lysates from E. coli[pGPl-21 with plasmid pT7-6 alone. Stn denotes expression of the stn* gene from E. co[i[pPE49]. Gst::Stn represents fusion polypeptide from E. coli[pGEXstn*] TrxA::Stn denote fusion

or E. coli[pGEXstn]. TrxA::Stn* and polypeptides produced by E. coli[pTRXFUSstn*] and E. coli[pTRXFUSstn], respectively. The indicated concentration of NaCl was added during growth of E. coli[pTRXFUSstn]. Cell lysates from E. coli[pTRXFUS] with and without NaCl were used as controls. Average values from three independent experiments were plotted.

85

50-fold increase in solubility of the TrxA::Stn antigen was noted as determined by ELISA. It is worth mentioning that the biological activity of TrxA::Stn from cultures grown with high osmolarity was not increased in proportion to the increase in the Stn ELISA. CHO cell elongation titers increased approx. twofold when the stn and stn* genes were expressed in high-osmolarity medium, compared to cells grown in LB medium (Fig. 5). This disparity could be due to differences in the sensitivity of the two assay systems or to degradation of Stn (Fig. 3B, lanes 4, 5).

(d) Conclusions (I) We increased the expression of the stn gene over

that obtained with the T7 expression system by using gene fusion expression systems, such as pGEX-KG and pTRXFUS. (2) Most of the Stn antigen was insoluble, using either of the fusion vector systems; however, its solubility increased when fused with TrxA, and this resulted in enhanced biological activity. (3) Expression of stn was positively regulated by NaCl, which is known to affect DNA superhelicity.

ACKNOWLEDGEMENTS

Vector pGEX-KG was obtained from Dr. J.E. Dixon, Purdue University, Indiana. The newly described vector system (pTRXFUS) was procured from Dr. E.R. LaVallie, Genetics Institute, Cambridge, MA, USA. This study was supported by research grant ROl AI 18401 from the National Institutes of Health.

REFERENCES Andersen-Beckh, B., Binz, T, Kurazono, H, Mayer, T, Eisel, U. and Niemann, H.: Expression of tetanus toxin subfragments in vitro and characterization of epitopes. Infect. Immun. 57 (1989) 3498-3505. Chopra, A.K., Peterson, J.W., Chary, P. and Prasad, R.: Molecular characterization of an enterotoxin from Salmonella ryphimurium. Microbial Pathol. 16 (1994) in press. Chary, P., Prasad, R, Chopra, A.K. and Peterson, J.W.: Location of the enterotoxin gene from Salmonella typhimurium and characterization of the gene products. FEMS Microbial. Lett. 111 (1993) 87-92. Galan, J.E. and Curtiss III, R.: Expression of Salmonella typhimurium genes required for invasion is regulated by changes in DNA supercoiling. Infect. Immun. 58 (1990) 1879-1885. Guan, K. and Dixon, J.E. Eukaryotic proteins expressed in E. coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. Anal. Biochem. 192 (1991) 262-267. Groisman, E.A., Fields, PI. and Heffron, F.: Molecular biology of Salmonella pathogenesis. In: Iglewski, B.H. and Clark, V.L. (Eds.), Molecular Basis of Bacterial Pathogenesis. Academic Press, San Diego, CA, 1990, pp. 251-267. Laemmli, U.K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227 (1970) 680-685. LaValhe, E.R., DiBlasio, E.A., Kovacic, S., Grant, K.L., Schendel, P.F. and McCoy, J.M.: A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm. Biotechnology. 11 (1993) 1877193. Popoff, M.Y.: Virulence-associated factors of Salmonella: from molecular biology to diagnosis. Bull. Acad. Natl. Med. 175 (1991) 811-821. Prasad, R., Chopra, A.K., Chary, P. and Peterson, J.W.: Expression and characterization of the cloned Salmonella typhimurium enterotoxin. Microbial Pathol. 13 (1992) 109-121. Prasad, R., Chopra, A.K., Peterson, J.W., Pericas, R. and Houston, C.W.: Biological and immunological characterization of a cloned cholera toxin-like enterotoxin from Salmonella typhimurium. Microbial Pathol. 9 (1990) 3155329. Sanger, F., Nicklen, S. and Coulson, A.R.: DNA sequencing with chainterminating inhibitors. Proc. Nat]. Acad. Sci. USA 74 (1977) 5463-5467. Tabor, S. and Richardson, C.C.: A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc. Natl. Acad. Sci. USA 82 (1985) 1074-1078.