Expression and Characterization of Group AStreptococcus Extracellular Cysteine Protease Recombinant Mutant Proteins and Documentation of Seroconversion during Human Invasive Disease Episodes

Bacterial strains and plasmids.The His tag expression vector pPROEX-1 (Gibco-BRL/Life Technologies, Grand Island, N.Y.) was used to construct plasmids for production of recombinant proteins. pSEBC2S is a derivative of plasmid pCR-Script AmpSK(+) (Stratagene, La Jolla, Calif.) that contains the speB gene with a mutant codon 192 that converts the active-site Cys to Ser (22). The plasmid contains the entire speB coding region plus 160 bp of upstream noncoding DNA (10). Escherichia coliDH5α and BL21 (a protease-deficient organism) were purchased from Gibco-BRL/Life Technologies and Stratagene, respectively. Additional molecular biology and immunology reagents were purchased from Gibco-BRL/Life Technologies, Sigma Chemical Co. (St. Louis, Mo.), or Bio-Rad (Richmond, Calif.).

Construction of vectors for expression of mutant proteins.The entire speB coding region was amplified from plasmid pSEBC2S (Fig. 1) with primers SG1 (5′-GCCCATATGAATAAAAAGAAATTAGGTATC-3′) and SG2 (5′-CGTAGGCCTCGTGCCTCAGGTTCTGTTCTA-3′) and the GenAmp PCR kit (Perkin-Elmer, Branchburg, N.J.). The PCR amplifications were performed with a Perkin-Elmer 9600 thermal cycler with Pfupolymerase (Stratagene), and the following parameters were used: 30 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 45 s, and extension at 72°C for 90 s. The reaction conditions included a 5-min incubation at 94°C before the initial amplification and a 5-min final incubation at 72°C after the 30 cycles were complete.

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Fig. 1.

Construction of SpeB expression plasmids. The vectors are based on the speB gene of MGAS 1719 (15). This speB allele was previously subjected to site-specific mutagenesis to create a gene that produces a C192S mutant SpeB molecule (22). The entire open reading frame of the mutant gene was amplified by PCR from pSEBC2S (22) and cloned into the multiple-cloning site of the His-tag expression vector pPROEX-1 to yield pGM-1. pGM-5 is identical to pGM-1 except that it lacks an 81-bp segment encoding the 27-amino-acid (aa) secretion signal sequence. pGM-8, made by cloning speB codons 146 to 398 into pPROEX-1, expresses a 28-kDa mature form of SpeB containing the C192S amino acid substitution. pGM-6 was constructed by cloning speB codons 28 to 145 (encoding the propeptide fragment) into pPROEX-1. SP, signal peptide; PP, propeptide; MP, mature protein.

The amplified speB gene was then cloned into pPROEX-1, and the plasmid was used to transform E. coli DH5α. Following selection on Luria-Bertani medium plates containing ampicillin (100 μg/ml), colonies were screened for the presence of the desired recombinant construct by digestion with NdeI andStuI. Automated DNA sequence analysis (15) documented that the speB gene was in the correct reading frame and contained no spurious mutations. Because this plasmid (pGM-1) had an 81-bp segment encoding the 27-amino-acid secretion signal sequence, a vector that contained speB lacking this leader sequence was constructed. To construct this vector (pGM-5), thespeB gene lacking the unwanted 81-bp region was amplified from pGM-1 with primers SG13 (5′-GGGGGCGCCGATCAAAACTTTGCTCGT-3′) and SG2 (Fig. 1). The amplified fragment was cloned into pPROEX-1, and ampicillin-resistant colonies were screened by PCR for the presence of speB. A colony that had speB in the correct orientation was identified. Automated DNA sequence analysis confirmed the correct reading frame and lack of spurious mutations.

Analogous strategies were used to construct plasmids with fragments of the speB gene containing codons 146 to 398 (pGM-8) and 28 to 145 (pGM-6). pGM-8 is designed to express a 28-kDa protein corresponding to the mature form of SpeB, and pGM-6 should express a 12-kDa molecule corresponding to the SpeB propeptide. pGM-6 was constructed from a speB PCR product generated with primers SG13 and SG14 (5′-CGTAGGCCTCTATTTAATCTCAGCGGTA-3′), and primers SG15 (5′-GGGGGCGCCCAACCAGTTGTTAAATCTCTCCT-3′) and SG2 were used to make the amplification product used for pGM-8 (Fig.1). All DNA inserts were sequenced in their entirety to confirm that no undesired mutations were present.

Expression and purification of recombinant proteins.Recombinant proteins were obtained from E. coli BL21 containing the appropriate plasmids after induction with isopropyl-β-d-thiogalactopyranoside (IPTG). Cells were grown overnight at 30°C, diluted 1:100 in Luria-Bertani medium supplemented with 100 μg of ampicillin per ml, and cultured to an optical density at 590 nm of 0.5 to 1.0. IPTG was added (final concentration, 0.6 mM), and the cells were grown for an additional 2 to 6 h. After centrifugation at 10,000 × g for 10 min, the supernatant was discarded and the cell pellet was stored at −70°C. The cells were resuspended in 5 volumes of 50 mM Tris-HCl (pH 8.5) containing 10 mM 2-mercaptoethanol and 1 mM phenylmethylsulfonyl fluoride and lysed by sonication with three 20-s pulses generated by a Sonifier Cell Disrupter 250 (Branson, Danbury, Conn.). After each 20-s burst, the cells were cooled for 5 min in a wet-ice bath. Cell wall debris was removed by centrifugation at 10,000 × g for 10 min, and the supernatant was applied to a 1- to 5-ml column of Ni-nitrilotriacetic acid resin (Qiagen, Chatsworth, Calif.) equilibrated by being washed with 5 volumes of buffer A (20 mM Tris-HCl [pH 8.5], 100 mM KCl, 20 mM imidazole, 10 mM 2-mercaptoethanol, 10% [vol/vol] glycerol). The column was washed with 10 volumes of buffer A, 2 volumes of buffer B (20 mM Tris-HCl [pH 8.5], 1 M KCl, 10 mM 2-mercaptoethanol, 10% [vol/vol] glycerol), and 2 volumes of buffer A to remove unbound protein. Recombinant protein containing the His tag was eluted from the column by washing with 5 volumes of buffer C (20 mM Tris-HCl [pH 8.5], 100 mM KCl, 100 mM imidazole, 10 mM 2-mercaptoethanol, 10% [vol/vol] glycerol). Recombinant proteins without the His tag were purified after direct cleavage of the His tag from the column-bound protein by treatment with approximately 1,000 U of recombinant tobacco etch virus (rTEV) protease per 3 mg of bound fusion protein. When this procedure was used, the rTEV protease was added to 5 ml of buffer A supplemented with 1 mM dithiothreitol and 0.5 mM EDTA and digestion was conducted for 2 h at 30°C on a shaking platform.

The recovered proteins were concentrated to 0.5 ml with Centrisep 3 or Centrisep 10 concentrators (Amicon, Beverly, Mass.), reconstituted to 3.0 ml with phosphate-buffered saline (PBS), and desalted either with desalting columns (Econopac 10 DG columns; Bio-Rad) or by dialysis against PBS with Slide-A-Lyser cassettes (Pierce, Rockford, Ill.). The concentration of the purified protein was estimated by a protein assay (Pierce), and the material was stored at 4°C. The purity of recombinant proteins was estimated by silver staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Purification of native SpeB from strain MGAS 1719.Native 28-kDa SpeB protease was purified from the culture supernatant of strain MGAS 1719 as described previously (14). The purified protein was >95% pure, as analyzed by SDS-PAGE and staining with Coomassie brilliant blue.

Amino-terminal sequencing of recombinant proteins.A 20-μg portion of each recombinant protein with the His tag removed by digestion with rTEV protease was analyzed by SDS-PAGE, transferred to a Problott membrane (Applied Biosystems, Foster City, Calif.), and stained with Coomassie brilliant blue. The desired protein band was excised and analyzed with an Applied Biosystems model 473A protein sequencer located at the Baylor College of Medicine Core Protein Facility.

SDS-PAGE and Western blot analysis.Standard SDS-PAGE procedures were used to test for the expression of recombinant proteins and to assess their purity. After SDS-PAGE, the gel was rinsed with deionized water and the proteins were transferred to a nitrocellulose membrane (Bio-Rad) with transfer buffer. The membrane was incubated for 1 h with 0.5% blocking agent (Amersham) and then for 1 h with TBS (20 mM Tris, 500 mM NaCl [pH 7.5]) containing 1% gelatin and primary antibody. The primary antibody used was mouse monoclonal antibody 2A3-B2-C12 (21) (1:200 dilution), rabbit polyclonal antibody raised against purified C192S zymogen (1:5,000 dilution), or human convalescent-phase sera (1:500 dilution) obtained from patients with invasive GAS infections. All serum dilutions were made with TBS containing 1% gelatin. The membranes were then washed with 0.1% Tween–TBS and water for 10 min each and incubated with secondary antibody (1:2,000 dilution of goat antibody conjugated to horseradish peroxidase [Bio-Rad]) diluted in TBS containing 1% gelatin. The membranes were washed with 0.1% Tween–TBS and water for 10 min each, and the antibody-antigen interaction was visualized with Bio-Rad developing reagent. The reaction was terminated with deionized water.

Rabbit polyclonal antisera.Polyclonal antisera were raised against the SpeB C192S zymogen and propeptide by immunizing rabbits with recombinant proteins purified to apparent homogeneity. The His tag was removed by digestion with rTEV protease before the animals were immunized. The sera were made under contract by standard procedures used by the supplier (Bethyl Laboratories, Montgomery, Tex.).

Streptococcal protease assays.Four assays were used to test for SpeB proteolytic activity. The streptococcal protease assay used routinely during purification is based on the ability of the enzyme to cleave bovine casein embedded in an agarose gel matrix (22). A second assay exploits the capability of streptococcal protease to cleave human fibronectin and has been described previously (22). To test for the ability of active wild-type cysteine protease to process recombinant C192S zymogen, active protease was incubated at 37°C for 30 min to 2 h with purified C192S zymogen at a molar ratio of 1:1 or 1:10 (22). The samples were analyzed by SDS-PAGE, and the products were visualized by silver staining. To test for the ability of active cysteine protease to degrade the recombinant SpeB propeptide, active wild-type protease was incubated at 37°C for 2 h with purified SpeB propeptide at a molar ratio of 1:1. The samples were analyzed by SDS-PAGE, and the products were visualized in parallel by staining with Coomassie brilliant blue or Western analysis with rabbit anti-propeptide.

Immuno-dot blot analysis of human patient sera.To test for human patient antibodies directed against the purified C192S zymogen, 1.0 μg of purified recombinant protein was applied to a nitrocellulose membrane. The blot was dried briefly in air, and the membrane was incubated with 0.5% blocking agent (Amersham) for 1 h. Acute- and convalescent-phase sera were diluted 1:500 and used as the primary antibody. After 1 h of incubation with primary antibody, the blots were washed with 0.1% Tween–TBS and water for 10 min each and incubated with goat anti-human secondary antibody (1:2,000 dilution of goat antibody conjugated with horseradish peroxidase diluted in TBS containing 1% gelatin). The membranes were then washed with 0.1% Tween–TBS and water for 10 min each, and the antibody-antigen interaction was visualized with Bio-Rad developing reagent. The reaction was terminated with deionized water.

Measurement of human anti-SpeB antibodies in serum by ELISA.Microtiter plates (Immulon 1; Dynatech Laboratories, Inc., Chantilly, Va.) were coated with 100 μl of a solution of test antigen (5 μg/ml in carbonate buffer [pH 9.6]). The plates were incubated overnight at room temperature and washed three times with 0.1 ml of PBS containing 0.1% Tween 20 adjusted to pH 7.4 (TPBS). The plates were blocked for 30 min at 37°C with 5% gelatin in TPBS, washed three times with TPBS, and incubated for 2 h at 37°C with 50 μl of serum samples diluted in TPBS. After three washes with TPBS, the plates were probed for 2 h at 37°C with 50 μl of horseradish peroxidase-conjugated goat anti-human immunoglobulin G (heavy and light chains) (Bio-Rad) diluted 1:2,000 in TPBS. After a further three washes, the plates were incubated with 2,2′-azino-di-[3-ethylbenzthiazoline sulfonate (6)] (ABTS; Boehringer Mannheim) as the development agent for 20 min at room temperature in the dark. The reaction was terminated by adding 20 μl of 1.75% SDS solution. The absorbance was measured at 405 nm (Microplate Autoreader EL311; Bio-Tek Instruments, Inc., Winooski, Vt.), and geometric mean titers were calculated. All samples were assayed in triplicate.