High level expression of Streptococcus pyogenes erythrogenic toxin A (SPE A) in Escherichia coli and its rapid purification by HPLC

MICROBIOLOGY LETTERS

ELSEVIER

FEMS Microbiology

Letters

I32 (I 995) 209-2 I3

High level expression of Streptococcus pyogenes erythrogenic toxin A ( SPE A) in Escherichia coli and its rapid purification by HPLC Mitsuyo Yamamoto, Joseph J. Ferretti Received

*

I I July 1995: revised I4 August 1995: accepted II August 1995

Abstract The speA gene encoding streptococcal erythrogenic toxin A (SPE A) from Sfrepfococcus p~~~grnrs bacteriophage T12 was overexpressed in Escherichiu coli under the control of the T7 promoter. Since most of the expressed protein was found in the periplasmic space, an osmotic shock extraction with 0.5 M sucrose resulted in a highly enriched preparation of SPE A. An additional two-step purification employing high pressure liquid chromatography resulted in a purified SPE A protein. Keworr/.s:

S/r~pfoc,oc,c,~rspygew.s:

Erythrogenic

toxin A; Purification:

1. Introduction The production of streptococcal erythrogenic toxin A (SPE A), also known as streptococcal pyrogenic exotoxin, by certain strains of Streptococcus pygenes has been of great interest because of its suggested role in streptococcal diseases [I]. This extracellular protein is produced only by lysogenic strains of group A streptococci [2], the gene has been cloned and shown to reside on a bacteriophage chromosome [3,4], and sequence analysis has shown that it has a high degree of similarity to staphylococcal enterotoxins [5]. SPE A was first described as the toxin responsible for the rash of scarlet fever [6] and subsequently a variety of activities have been associated with it [I]. including its ability to serve as a

’ Corresponding author. Tel.: + I (40.5) 271 2133 Fax: (40.5) 27 I 31 17: E-mail: [email protected] 0378.1097/9.5/$09.50 0 1995 Federation SSDl 037% lOY7(95)003 12-6

of European

+ I

Microbiological

High pressure liquid chromatography

superantigen and stimulate T cells to release lymphokines [7,8]. The ability to obtain highly purified preparations of SPE A is crucial for studies on its biological activity and in this report we present a rapid procedure employing high pressure liquid chromatography (HPLC) to obtain purified SPE A from overexpressed cultures of Escherichia co/i containing the speA gene.

2. Materials

and methods

2.1. Cloning i’ector

of speA in au E. coli

A phage ligated sesses

or ~erexpression

I .8-kb EcoRI-Sal1 fragment from bacterioT12 known to contain the speA gene [3] was into vector pET23( + 1 (Novagen) which posthe T7 promoter and this construct was trans-

Societies. All rights reserved

210

M. Yamamoto. J.J. Ferrrtti/

FEMS Microhiolog~

formed into E. coli HMS174 (Novagen). Following extraction of plasmid DNA (pET23( +) + 1.8 kb .vpeA fragment is designated pMY I>, it was then transformed into E. co/i BL2 1(DE3) (Novagen). SPE A production of the transformants was monitored by Ouchterlony immunodiffusion (bacterial cells were lysed in 25 mM Tris . HCl pH 8.0 containing 0.9% glucose, 2 mM EDTA pH 8.0 and 1% SDS> with rabbit antiserum specific to SPE A. 2.2. Osmotic shock extraction Strain BL2 l(DE3, pMY I) was grown in 200 ml M9ZB medium [9] containing 50 pg of ampicillin per ml. When the culture reached an OD,,, of 0.6, 0.5 mM IPTG was added to induce the T7 RNA polymerase, present in plasmid DE3 and under the control of 1acP. The culture was incubated another 2 h, after which the cells were pelleted by centrifugation (8000 X g, 10 min), resuspended in l/l0 volume of ice-cold spheroplast buffer (100 mM Tris . HCI pH 8.0, 0.5 M sucrose, 1 mM EDTA) and left on ice for IO min. The supematant (sucrose fraction) was isolated by centrifugation (8000 X s, IO min) and the cell pellets were resuspended in the same volume of ice-cold water. After 30 min on ice, 1 mM MgCl was added and the cell suspension was left on ice for another 30 min. The supematant (water fraction) was saved after centrifugation. 2.3. High pressure

liquid chromatography

Lettrrs

132 (199.7) 209-213

containing < 1 mg of protein, was applied to the column and eluted with a 15-min linear gradient from 100% buffer A (10 mM sodium acetate buffer pH 4.0, 1 mM EDTA) to 100% buffer B (10 mM sodium acetate buffer pH 4.0, 1 mM EDTA, 1 M NaCl). 2.4. Polyacrylamide

gel electrophoresis

(PAGE)

Proteins for electrophoresis were solubilized and loaded onto a 10% sodium dodecyl sulfate-polyacrylamide gel using the buffer system of Laemmli [IO]. PAGE was performed and the protein bands stained with Coomassie brilliant blue R or silver stain (Bio-Rad). 2.5. Proliferative phocytes (PBLI

response of peripheral

blood lym-

Human PBMC (lO’/well) in serum-free HB 101 (Urbem Scientific Inc.), containing Polymyxin B per ml, were stimulated with fied SPE A. The cultures were incubated after which they were pulsed with 3H-TdR

medium 50 pg of the purifor 48 h, for 18 h.

(HPLCI

The sucrose fraction described above was dialysed against 10 mM Tris . HCI (pH 8.0) containing 1 mM EDTA and concentrated by ultrafiltration (UHP76, Microfiltration system Co.> approximately to l/20 the original volume. The sample, containing less than 5 mg of protein in about 2 ml, was applied to a MA7Q anion exchange column (50 X 7.8 mm, Bio-Rad Lab.) and was eluted with a 15-min linear gradient from 100% buffer A (10 mM Tris . HCl pH 8.0, 1 mM EDTA) to 100% buffer B ( 10 mM Tris. HCI pH 8.0, 1 mM EDTA, I M NaCl). The solvent flow rate was 2 ml min- ‘. For a final purification step, a MA7S (30 X 4.6 mm, Bio-Rad Lab.) cation exchange column was prepared with 4 ml of 10 mM sodium acetate buffer (pH 4.01, containing 1 mM EDTA. The sample,

SPEA-

Fig. I. SDS-PAGE of SPE A-containing samples stained with Coomassie brilliant blue R from (lane I) osmotic-shock extraction of periplasmic proteins and (lane 2) water lysis of cells from E. coli BL21(DE3, pMYI). The major protein band was found at about 25 kDa, as determined by comparison with molecular size markers, and identified as SPE A by Western immunoblot (data not shown).

M. Ymnamoro, J.J. Ferrerri/ FEMS Micmhidog~

211

L&revs 132 ( IYY5)2OY-2 13

1

-I 0.0

..I‘.

I

..,

-1

1

I

.I,,.

I

I

I

I

1

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25.0 WI-S

The sample was Fig. 2 Elution pattern of the osmotic-shock extract of periplasmic proteins on an HPLC anion exchange column(MA7Q) eluted with a IS-min linear gradient from 100% buffer A (IO mM Tris HCI pH 8.0. I mM EDTA) to 100% buffer B (buffer A + I M NaCI).

3. Results and discussion An osmotic-shock extraction procedure was employed to obtain the crude SPE A following overexpression. Samples of both the sucrose and water fraction (10 ml) were analysed by SDS-PAGE and the presence of SPE A confirmed by Western immunoblot (data not shown). As shown in Fig. I, the sucrose fraction (lane 1) contained primarily SPE A with little other protein, while the water fraction contained a large number of proteins as well as SPE A. No protein was observed at this position with a

sample from a culture with the same vector but no speA insert (data not shown). Further purification of the SPE A found in the sucrose fraction was achieved by HPLC. Fig. 2 shows a typical elution pattern from an MA7Q column. The largest peak, which eluted at about 50% of buffer B, contained most of the SPE A as detected by SDS-PAGE and a 4-5-ml sample was collected. Several minor bands were also observed, similar to those obtained in lane 1 of Fig. 1. The sample was desalted and concentrated with Centricon- IO (Amicon Inc.) to a volume of about 500 ~1.

0 d

I 0.0

I

,

I

,

I

I

I

1

20 MINUTES

Fig. 3. Elution pattern of SPE A protein fraction obtained from the anion exchange column and applied to an HPLC cation exchange column (MA7S). The sample was eluted with 15.min linear gradient from 100% buffer A (IO mM sodium acetate pH 4.0. I mM EDTA) to 100% buffer B (buffer A + I M NaCI). Inset shows a silver-stained SDS-PAGE of the eluted samples which contain SPE A.

212

*‘

0

0.1

1

10

100

ng WEAl ml Fig. 4. Proliferative response of human PBL to two indepently purified samples of SPE A.

For a final purification step, the above sample was applied to a MA7S (30 X 4.6 mm, Bio-Rad Lab.) cation exchange column. Two peaks containing SPEA were eluted between 2.5% and 60% of the buffer B area (Fig. 3). The samples from both peaks showed a single band of SPE A as ascertained by silver-stained 10% SDS-PAGE, although the valley region between the two peaks sometimes contained a small amount of other protein which was barely detectable in the silver-stained gel (arrow, Fig. 3). The fractions from areas I and 3 of Fig. 3 were combined, and together represented more than 80% of the protein originally placed on the column. Approximately I mg of pure SPE A was obtained from a l-l culture of cells. To test the biological activity of the purified SPE A, use was made of the proliferative response of peripheral blood lymphocytes (PBL) to SPE A. As shown in Fig. 4, 1 ng of SPEA per ml resulted in a significant stimulation of human PBLs. The purity of SPE A in various test assays is an important consideration in the evaluation of its biological activity. For example, SPE A is one of several streptococcal proteins which act as a superantigen to stimulate T lymphocyte production of lymphokines [7,8]. However, there has not been agreement about the specificities of T cell stimulation by SPE A 18.1 1,121, perhaps because trace amounts of other proteins produced by S. p~ogerzes can have activities which mask or interfere with the

activities attributed to SPE A. Purification of SPE A as described in this report excludes interference by other streptococcal products since the .ywA gene is the only streptococcal gene expressed by E. coli. The presence of a signal peptide on the unprocessed SPE A allows its accumulation in the periplasmic space of E. coli and, when overexpressed, represents the bulk of protein obtained by osmotic shock extraction. An easy two-step application of this protein to HPLC columns allows separation and recovery of a highly purified and biologically active SPE A suitable for further analysis.

Acknowledgements We thank Rodney Tweten and Jimmy Ballard for assistance with the HPLC purification, and Norma McElwee for performing the proliferation assays. This research was supported by Public Health Service grant AI 19304 from the National Institutes of Health.

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