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• W1968839949 abstract "Streptopain is a cysteine protease expressed by Streptococcus pyogenes. To study the maturation mechanism of streptopain, wild-type and Q186N, C192S, H340R, N356D and W357A mutant proteins were expressed in Escherichia coli and purified to homogeneity. Proteolytic analyses showed that the maturation of prostreptococcal pyrogenic exotoxin B zymogen (pro-SPE B) involves eight intermediates with a combination ofcis- and trans-processing. Based on the sequences of these intermediates, the substrate specificity of streptopain favors a hydrophobic residue at the P2 site. The relative autocatalytic rates of these mutants exhibited the order Q186N > W357A > N356D, C192S, H340R. Interestingly, the N356D mutant containing protease activity could not be converted into the 28-kDa form by autoprocessing. This observation suggested that Asn356 might involve the cis-processing of the propeptide. In addition, the maturation rates of pro-SPE B with trypsin and plasmin were 10- and 60-fold slower than that with active mature streptopain. These findings indicate that active mature streptopain likely plays the most important role in the maturation of pro-SPE B under physiological conditions. Streptopain is a cysteine protease expressed by Streptococcus pyogenes. To study the maturation mechanism of streptopain, wild-type and Q186N, C192S, H340R, N356D and W357A mutant proteins were expressed in Escherichia coli and purified to homogeneity. Proteolytic analyses showed that the maturation of prostreptococcal pyrogenic exotoxin B zymogen (pro-SPE B) involves eight intermediates with a combination ofcis- and trans-processing. Based on the sequences of these intermediates, the substrate specificity of streptopain favors a hydrophobic residue at the P2 site. The relative autocatalytic rates of these mutants exhibited the order Q186N > W357A > N356D, C192S, H340R. Interestingly, the N356D mutant containing protease activity could not be converted into the 28-kDa form by autoprocessing. This observation suggested that Asn356 might involve the cis-processing of the propeptide. In addition, the maturation rates of pro-SPE B with trypsin and plasmin were 10- and 60-fold slower than that with active mature streptopain. These findings indicate that active mature streptopain likely plays the most important role in the maturation of pro-SPE B under physiological conditions. group A streptococcus streptococcal pyrogenic exotoxin B phosphate-buffered saline dithiothreitol Group A streptococcus (GAS)1 causes several human diseases, including as pharyngitis, acute rheumatic fever, scarlet fever, post-streptococcal glomerulonephritis, and toxic shock-like syndrome (1Hoge C.W. Schwartz B. Talkington D.F. Breiman R.F. MacNeill E.M. Englender S.J. J. Am. Med. Assoc. 1993; 269: 384-389Crossref PubMed Scopus (373) Google Scholar, 2Molinari G. Cursharan S.C. Curr. Opin. Microbiol. 1999; 2: 56-61Crossref PubMed Scopus (55) Google Scholar). Virtually all strains of GAS isolated from patients with invasive disease express an extracellular cysteine protease known as streptopain (EC 3.4.22.10), with synonyms including streptococcal pyrogenic exotoxin B (SPE B or SpeB), streptococcus peptidase A, and streptococcal cysteine protease (3Bisno A.L. N. Engl. J. Med. 1991; 325: 783-793Crossref PubMed Scopus (381) Google Scholar, 4Gubba S. Low D.E. Musser J.M. Infect. Immun. 1998; 66: 765-770Crossref PubMed Google Scholar, 5Tai J.Y. Kortt A.A. Liu T.Y. Elliott S.D. J. Biol. Chem. 1976; 251: 1955-1959Abstract Full Text PDF PubMed Google Scholar). Many reports also suggest that streptopain is an important virulence factor in streptococcal infections (6Burns E.H. Lukomski Jr., S. Rurangirwa J. Podbielski A. Musser J.M. Microb. Pathog. 1998; 24: 333-339Crossref PubMed Scopus (34) Google Scholar, 7Kazmi S.U. Kansal R. Aziz R.K. Hooshdaran M. Norrby-Teglund A. Low D.E. Halim A.B. Kotb M. Infect. Immun. 2001; 69: 4988-4995Crossref PubMed Scopus (68) Google Scholar, 8Kuo C.-F. Wu J.-J. Lin K.Y. Tsai P.J. Lee S.C. Jin Y.T. Lei H.Y. Lin Y.-S. Infect. Immun. 1998; 66: 3931-3935Crossref PubMed Google Scholar, 9Kuo C.-F. Wu J.-J. Tsai P.J. Kao F.J. Lei H.Y. Lin M.T. Lin Y.-S. Infect. Immun. 1999; 67: 126-130Crossref PubMed Google Scholar, 10Lukomski S. Montgomery C.A. Rurangirwa J. Geske R.S. Barrish J.P. Adams G.J. Musser J.M. Infect. Immun. 1999; 67: 1779-1788Crossref PubMed Google Scholar, 11Ohara-Nemoto Y. Sasaki M. Kaneko M. Nemoto T. Ota M. Can. J. Microbiol. 1994; 40: 930-936Crossref PubMed Scopus (33) Google Scholar). Streptopain produced from GAS is released extracellularly to culture medium as a zymogen (pro-SPE B) with a molecular mass of 40-kDa, and the active form is a 28-kDa mature protease (12Hauser A.R. Schlievert P.M. J. Bacteriol. 1990; 172: 4536-4542Crossref PubMed Scopus (116) Google Scholar, 13Kapur V. Topouzis S. Majesky M.W. Li L.L. Hamrick M.R. Hamill R.J. Patti J.M. Musser J.M. Microb. Pathog. 1993; 15: 327-346Crossref PubMed Scopus (187) Google Scholar, 14Liu T.-Y. Elliott S.D. J. Biol. Chem. 1965; 240: 1138-1142Abstract Full Text PDF PubMed Google Scholar, 15Tsai P.J. Kuo C.-F. Lin K.Y. Lin Y.-S. Lei H.Y. Chen F.F. Wang J.R. Wu J.-J. Infect. Immun. 1998; 66: 1460-1466Crossref PubMed Google Scholar, 16Nomizu M. Pietrzynski G. Kato T. Lachance P. Menard R. Ziomek E. J. Biol. Chem. 2001; 276: 44551-44556Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Because the 28-kDa active form of streptopain plays an important role in GAS pathogenesis, studies on the maturation of pro-SPE B have become a subject of interest (12Hauser A.R. Schlievert P.M. J. Bacteriol. 1990; 172: 4536-4542Crossref PubMed Scopus (116) Google Scholar, 14Liu T.-Y. Elliott S.D. J. Biol. Chem. 1965; 240: 1138-1142Abstract Full Text PDF PubMed Google Scholar, 15Tsai P.J. Kuo C.-F. Lin K.Y. Lin Y.-S. Lei H.Y. Chen F.F. Wang J.R. Wu J.-J. Infect. Immun. 1998; 66: 1460-1466Crossref PubMed Google Scholar,17Doran J.D. Nomizu M. Takebe S. Menard R. Griffith D. Ziomek E. Eur. J. Biochem. 1999; 263: 145-151Crossref PubMed Scopus (55) Google Scholar). Zymogen activation produces a prompt and irreversible response to a physiological stimulus, and it is capable of initiating new physiological functions. The maturation of a zymogen involves limited proteolysis and can be finished either in a single activation step or in a consecutive cascade. The maturation of pro-SPE B can be achieved under a variety of conditions, including proteolysis by autoprocessing, trypsin, subtilisin, or the 28-kDa active form of streptopain (12Hauser A.R. Schlievert P.M. J. Bacteriol. 1990; 172: 4536-4542Crossref PubMed Scopus (116) Google Scholar, 14Liu T.-Y. Elliott S.D. J. Biol. Chem. 1965; 240: 1138-1142Abstract Full Text PDF PubMed Google Scholar,15Tsai P.J. Kuo C.-F. Lin K.Y. Lin Y.-S. Lei H.Y. Chen F.F. Wang J.R. Wu J.-J. Infect. Immun. 1998; 66: 1460-1466Crossref PubMed Google Scholar, 17Doran J.D. Nomizu M. Takebe S. Menard R. Griffith D. Ziomek E. Eur. J. Biochem. 1999; 263: 145-151Crossref PubMed Scopus (55) Google Scholar). Similar autoactivation processes have been found in other families of cysteine proteases; specifically, the maturation mechanism of propapain has been fully characterized (17Doran J.D. Nomizu M. Takebe S. Menard R. Griffith D. Ziomek E. Eur. J. Biochem. 1999; 263: 145-151Crossref PubMed Scopus (55) Google Scholar, 18Vernet T. Berti P.J. de Montigny C. Musil R. Tessier D.C. Menard R. Magny M.C. Storer A.C. Thomas D.Y. J. Biol. Chem. 1995; 270: 10838-10846Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 19Vernet T. Chatellier J. Tessier D.C. Thomas D.Y. Protein Eng. 1993; 6: 213-219Crossref PubMed Scopus (28) Google Scholar, 20Vernet T. Khouri H.E. Laflamme P. Tessier D.C. Musil R. Gour-Salin B.J. Storer A.C. Thomas D.Y. J. Biol. Chem. 1991; 266: 21451-21457Abstract Full Text PDF PubMed Google Scholar). Thecis-processing of propapain is characterized by a zero-order reaction, where the rate of propapain processing is independent of the propapain concentration. In contrast, the trans-processing of propapain is characterized by a higher order reaction, where the rate of propapain processing is dependent on the concentration of propapain (20Vernet T. Khouri H.E. Laflamme P. Tessier D.C. Musil R. Gour-Salin B.J. Storer A.C. Thomas D.Y. J. Biol. Chem. 1991; 266: 21451-21457Abstract Full Text PDF PubMed Google Scholar). In one recent study, investigators used multiple approaches to demonstrate that the maturation of pro-SPE B involves both cis- and trans-processing (17Doran J.D. Nomizu M. Takebe S. Menard R. Griffith D. Ziomek E. Eur. J. Biochem. 1999; 263: 145-151Crossref PubMed Scopus (55) Google Scholar). Pro-SPE B immobilized on a Sepharose resin is capable of liberating the 28-kDa form of streptopain from the column, indicating that the maturation of pro-SPE B involves cis-processing (17Doran J.D. Nomizu M. Takebe S. Menard R. Griffith D. Ziomek E. Eur. J. Biochem. 1999; 263: 145-151Crossref PubMed Scopus (55) Google Scholar). Although streptopain (C10) belongs to one of the cysteine protease families and undergoes similar maturation processing, it contains a prodomain different from that of other cysteine protease families (20Vernet T. Khouri H.E. Laflamme P. Tessier D.C. Musil R. Gour-Salin B.J. Storer A.C. Thomas D.Y. J. Biol. Chem. 1991; 266: 21451-21457Abstract Full Text PDF PubMed Google Scholar, 21Groves M.R. Coulombe R. Jenkins J. Cygler M. Proteins. 1998; 32: 504-514Crossref PubMed Scopus (75) Google Scholar). The maturation of pro-SPE B to the 28-kDa active form of streptopain can also be mediated by exogenous proteases such trypsin and subtilisin and by the active form of streptopain (14Liu T.-Y. Elliott S.D. J. Biol. Chem. 1965; 240: 1138-1142Abstract Full Text PDF PubMed Google Scholar). Streptopain is an extracellular plasmin-binding protein for nephritogenic streptococci (22Poon-King R. Bannan J. Viteri A. Cu G. Zabriskie J.B. J. Exp. Med. 1993; 178: 759-763Crossref PubMed Scopus (79) Google Scholar). The ability of streptopain to bind human plasminogen and plasmin may be important because these interactions may provide a means for GAS invasion. However, the maturation of pro-SPE B by exogenous proteases has never been fully addressed. To date, >60 families of proteases containing a cysteine residue at the active site have been found (21Groves M.R. Coulombe R. Jenkins J. Cygler M. Proteins. 1998; 32: 504-514Crossref PubMed Scopus (75) Google Scholar). According to the MEROPS Protease Database, 2Available at merops.sanger.ac.uk/. streptopain and papain belong to the C10 and C1 families of papain-like clan CA, respectively. Although there is only 207 identity and 437 similarity between the sequences of streptopain and papain, the catalytic residues of streptopain (Cys192, His340, and Asn356) occur in the same order as those in papain (Cys25, His159, and Asn175), with some identical nearby residues (5Tai J.Y. Kortt A.A. Liu T.Y. Elliott S.D. J. Biol. Chem. 1976; 251: 1955-1959Abstract Full Text PDF PubMed Google Scholar, 20Vernet T. Khouri H.E. Laflamme P. Tessier D.C. Musil R. Gour-Salin B.J. Storer A.C. Thomas D.Y. J. Biol. Chem. 1991; 266: 21451-21457Abstract Full Text PDF PubMed Google Scholar). However, little is known about the residues involved in the maturation processing of streptopain. For purposes of comparison with previous mutagenesis studies, in this study, we numbered the first methionine of the signal peptide of streptopain as position 1 (23Gubba S. Musser J.M. Infect. Immun. 2000; 68: 3716-3719Crossref PubMed Scopus (13) Google Scholar, 24Matsuka Y.V. Pillai S. Gubba S. Musser J.M. Olmsted S.B. Infect. Immun. 1999; 67: 4326-4333Crossref PubMed Google Scholar, 25Musser J.M. Stockbauer K. Kapur V. Rudgers G.W. Infect. Immun. 1996; 64: 1913-1917Crossref PubMed Google Scholar). To examine the kinetic and biochemical effects of mutation on the maturation processing of pro-SPE B, in this study, we expressed the wild-type protein and mutants Q186N, C192S, H340R, N356D, and W357A inEscherichia coli: Gln186 is the residue outside the substrate-binding pocket and contacts with the loop containing Cys192; Cys192 and His340 are the catalytic residues; and Asn356 and Trp357 are the residues near the active site of streptopain. Studies of the chemical modifications of cysteine, histidine, and tryptophan also indicate that these residues are essential for the protease activity of streptopain (5Tai J.Y. Kortt A.A. Liu T.Y. Elliott S.D. J. Biol. Chem. 1976; 251: 1955-1959Abstract Full Text PDF PubMed Google Scholar). Based on the x-ray structure of the streptopain C192S mutant (Fig. 1) (27Aiyar A. Xiang Y. Leis J. Methods Mol. Biol. 1996; 57: 177-191PubMed Google Scholar), the catalytic site of streptopain has a catalytic Cys-His dyad, which differs from most other cysteine proteases containing a Cys-His-Asn catalytic triad. Previous studies have shown that a mutation at Cys192 or His340 of streptopain leads to a complete loss of protease activity (4Gubba S. Low D.E. Musser J.M. Infect. Immun. 1998; 66: 765-770Crossref PubMed Google Scholar, 23Gubba S. Musser J.M. Infect. Immun. 2000; 68: 3716-3719Crossref PubMed Scopus (13) Google Scholar, 25Musser J.M. Stockbauer K. Kapur V. Rudgers G.W. Infect. Immun. 1996; 64: 1913-1917Crossref PubMed Google Scholar). Asn175 of papain is not essential for its protease activity, and it may play a role in orientating the catalytic residues. In streptopain, the carbonyl group of Trp357 may replace the function of Asn175 in papain (26Kagawa T.F. Cooney J.C. Baker H.M. McSweeney S. Liu M. Gubba S. Musser J.M. Baker E.N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2235-2240Crossref PubMed Scopus (93) Google Scholar). The crystal structure also demonstrates that Phe342, Trp357, and Phe367 form a hydrophobic pocket that fits a hydrophobic residue at the P2 site of the substrate (26Kagawa T.F. Cooney J.C. Baker H.M. McSweeney S. Liu M. Gubba S. Musser J.M. Baker E.N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2235-2240Crossref PubMed Scopus (93) Google Scholar). Although the role of Asn356 in streptopain is not clear, it is near the active-site region of streptopain. To compare the maturation of pro-SPE B and propapain, we applied the same approach used in propapain to study pro-SPE B (20Vernet T. Khouri H.E. Laflamme P. Tessier D.C. Musil R. Gour-Salin B.J. Storer A.C. Thomas D.Y. J. Biol. Chem. 1991; 266: 21451-21457Abstract Full Text PDF PubMed Google Scholar). We also investigated the processing of limited proteolysis of pro-SPE B by trypsin and plasmin and characterized the intermediates of pro-SPE B during its course of maturation. Our goal was to provide an additional basis for exploring the differences between autoproteolytic and proteolytic processing. We found that the maturation processing of pro-SPE B involves both cis- and trans-processing in a consecutive cascade with eight identifiable intermediates and that the 28-kDa active form of streptopain is the most effective protease for converting pro-SPE B into a mature protease throughtrans-processing. This study serves as the basis for gaining insight into streptococcal infections by exploring the maturation processing and characterization of streptopain. The genomic DNA of GAS was extracted from strain A20. The structural gene of pro-SPE B was amplified by PCR using the sense primer 5′-GGATCCGGATCCCATCATCATCATCATCATGATCAAAACTTTGCTCGTAACGAA-3′ with a His6 tag and BamHI recognition and by the antisense primer 5′-GGATCCGGATCCCTAAGGTTTGATGCCTACAACAG-3′ with BamHI recognition. The PCR product was purified and then cloned into the BamHI site of the pET-21a vector. The recombinant plasmid was transformed into the E. coli BL21(DE3) pLys strain, and the system was under the control of a strong T7 promoter. The wild-type construct was used to produce Q186N, C192S, H340R, N356D, and W357A mutations using overlap extension PCR (27Aiyar A. Xiang Y. Leis J. Methods Mol. Biol. 1996; 57: 177-191PubMed Google Scholar). Cells were grown at 37 °C for 6–8 h in LB medium (1 liter of 10 g of Bacto-Tryptone, 5 g of Bacto-yeast extract, and 10 g of NaCl) that was adjusted to pH 7.2 with 3n NaOH. The cells were cultured toA600 = 0.5–1.0. To the culture was added isopropyl-1-thio-औ-d-galactopyranoside (1 mm), and the culture was further incubated at 15–37 °C for 2–24 h to induce protein production. Cells were harvested by centrifugation and lysed by liquid shear with a French press to obtain the extract. To obtain soluble proteins, the conditions of protein expression were optimized by lowering the temperature and by varying induction periods. Under varying induction conditions, 10 ml of cells were collected by centrifugation and suspended in 1 ml of lysis buffer (20 mmTris-HCl, 200 mm NaCl, and 1 mg/ml lysozyme, pH 8.0). Each lysate was centrifuged at 10,000 × g for 10 min. The proteins in the supernatant (soluble) were collected, and the proteins in the pellet (insoluble) were dissolved in 1 ml of sample buffer. SDS-PAGE was performed to analyze the relative proportions of overexpressed proteins in the soluble and insoluble fractions. Recombinant wild-type streptopain was converted into a 28-kDa active enzyme during the course of purification. To prevent the conversion, mercuric chloride was added to the wild-type extract to a final concentration of 1 mm and was kept present throughout the purification. In addition, most of the expressed W357A mutant was present as an insoluble inclusion body, and a standard procedure using a denaturing condition was performed to refold the protein (41QIAGEN Inc. QIAGEN Manual. 2nd Ed. QIAGEN Inc., Chatsworth, CA1992Google Scholar). The inclusion body of the W357A mutant was solubilized in denaturing solution (4.5 m urea, 20 mm Tris-HCl, and 200 mm NaCl, pH 8.0), and the solution was diluted toA280 < 0.1. The protein was renatured by dialysis against 20 mm Tris-HCl and 200 mmNaCl, pH 8.0. The recombinant proteins were purified by Ni2+ chelating chromatography (Amersham Biosciences) with a gradient of 20–200 mm imidazole. The proteins were concentrated by Amicon ultrafiltration using a 10-kDa cutoff membrane and then exchanged with PBS. The final solutions were stored at −20 °C. Native streptopain was purified from strain A20 as previously described (8Kuo C.-F. Wu J.-J. Lin K.Y. Tsai P.J. Lee S.C. Jin Y.T. Lei H.Y. Lin Y.-S. Infect. Immun. 1998; 66: 3931-3935Crossref PubMed Google Scholar). One volume of 1 ml of bacterial culture was first grown overnight at 35 °C in 20 ml of TSBY medium (37 tryptic soy broth and 0.57 yeast extract). The culture was then added to 100 ml of TSBY medium. Streptopain was produced by growing cells at 37 °C for 22–24 h. The supernatant was collected by centrifugation and filtered through a 0.45-ॖm membrane filter. The filtrate was diluted with 400 ml of cold distilled water, and the pH was adjusted with 1n NaOH to 8.0. Then, 25 g of pre-equilibrated DEAE-Sepharose resin (Amersham Biosciences) with 20 mmTris-HCl, pH 8.0, were added to the filtrate. The solution was left for 30 min with occasional mixing, and the unbound protein was collected by filtration. The filtrate was concentrated to 100 ml by Amicon ultrafiltration using a 3-kDa cutoff membrane. The buffer was exchanged by ultrafiltration with 1 liter of 207 ethanol and 20 mmTris-HCl, pH 7.0. The final solution was loaded onto a Red A column (Dymatrex gel, Millipore Corp.). Streptopain was eluted using a linear gradient of 400 ml of 0–2 m NaCl with a flow rate of 20 ml/h, and 5-ml fractions were collected. SDS-PAGE showed that streptopain with a molecular mass of 28 kDa was homogeneous. The azocasein assay was used to test for proteolytic activity of streptopain and mutant proteins. The assay was modified as previously described (28Stockbauer K.E. Magoun L. Liu M. Burns Jr., E.H. Gubba S. Renish S. Pan X. Bodary S.C. Baker E. Coburn J. Leong J.M. Musser J.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 242-247Crossref PubMed Scopus (87) Google Scholar). Activity was determined by measuring the hydrolysis of azocasein based on the absorbance increase at 366 nm against time as described below. The reaction was initiated by addition of 20 ॖl of streptopain or mutant protein to 160 ॖl of reaction mixture containing 2.7 mg/ml azocasein, 5 mmdithiothreitol (DTT), and 5 mm EDTA (Sigma) in PBS. After incubating the solution at 37 °C for the designated time intervals ranging from 0 to 24 h, the reaction was stopped by addition of 40 ॖl of 157 ice-cold trichloroacetic acid. Absorbance was measured using a Beckman Model DU640 spectrophotometer. One enzyme unit is defined as the amount of protease required to release 1 ॖg of soluble azopeptide/min. The specific absorption coefficient (A366117 = 40) of the azocasein solution was calculated by measuring its absorption after total digestion (28Stockbauer K.E. Magoun L. Liu M. Burns Jr., E.H. Gubba S. Renish S. Pan X. Bodary S.C. Baker E. Coburn J. Leong J.M. Musser J.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 242-247Crossref PubMed Scopus (87) Google Scholar). Because the pro-SPE B C192S mutant does not exhibit any enzyme activity and exists as a 42-kDa zymogen, it can be used as the substrate for the 28-kDa active form of streptopain (17Doran J.D. Nomizu M. Takebe S. Menard R. Griffith D. Ziomek E. Eur. J. Biochem. 1999; 263: 145-151Crossref PubMed Scopus (55) Google Scholar). The reaction was carried out in a total of 20 ॖl of PBS containing 5 mm EDTA and 5 mm DTT. Purified or recombinant streptopain protein at a final concentration of 1.2 ॖmwas incubated with the 42-kDa C192S mutant (24 ॖm) at 37 °C for the designated time intervals ranging from 0 to 14 h. The reactions were quenched by addition of 5 ॖl of 50 ॖm E-64 (trans-epoxysuccinyl-l-leucylamido-(4-guanidino)butane) and incubated at 37 °C for 30 min. The solution was then heated at 100 °C for 10 min and analyzed by 127 SDS-PAGE. Gels were scanned using a Vilber Lourmat Model CN-TFX imaging system, and the intensities of the bands were integrated with BIO-1D Version 5.07 software. Proteolytic activity was measured as the disappearance of pro-SPE B, and relative reaction rates were obtained from the intensity change of the 42-kDa pro-SPE B C192S band against time. Plasmin and trypsin were obtained from Sigma. The reaction was carried out in a total of 20 ॖl of PBS containing 5 mm EDTA and 5 mm DTT. Plasmin or trypsin at a final concentration of 1.2 ॖm was incubated with the 42-kDa pro-SPE B C192S mutant (24 ॖm) at 37 °C for the designated time intervals ranging from 0 to 72 h. The reactions were quenched by addition of 5 ॖl of 5 mmphenylmethanesulfonyl fluoride and incubated at 37 °C for 30 min. The solution was then heated at 100 °C for 10 min and analyzed by 127 SDS-PAGE. The relative rates of processing were measured from the intensity change of the 42-kDa pro-SPE B C192S band against time. The reaction was carried out in PBS with 5 mm EDTA and 5 mm DTT. A total of 13.5 ॖg of pro-SPE B were incubated in volumes of 20, 35, 50, 75, 100, 200, 300, and 400 ॖl at 37 °C for the designated time intervals from 0 to 14 h. The final concentrations were 0.2, 0.35, 0.5, 0.75, 1, 2, 3, and 4 ॖm, respectively. The reaction was quenched by adding 5 ॖl of 50 ॖm E-64 and heating the solution at 100 °C for 10 min, and the solution was then lyophilized for gel analysis. Relative reaction rates were obtained from the intensity change of pro-SPE B against time. The molecular masses of the proteins were examined by mass spectrometry using a PerkinElmer Life Sciences triple quadrupole mass spectrometer (Model API365). Proteins were dissolved in 0.17 formic acid and 1007 methanol as the matrix. Streptopain, mutant proteins, and the cleavage products of wild-type and mutant proteins of pro-SPE B were analyzed by 127 SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membrane was stained with 0.27 Amido Black. The bands were excised and analyzed using an Applied Biosystems Model 477A Sequencer. CD spectroscopy was used to determine the secondary structures of wild-type and mutant proteins of pro-SPE B. CD spectra were measured at 27 °C on a Jasco J-720 spectropolarimeter that had been calibrated with camphosulfonic acid. Spectra were recorded under nitrogen between 185 and 260 nm and with a 1.0-nm spectral step size, a 1.0-nm bandwidth, and a 10 or 12 nm/min scan rate. The secondary structures of streptopain and its mutants were estimated using the convex constraint algorithm together with a least-square fitting program (LINCOMB) as described by Perczel et al. (29Perczel A. Hollosi M. Tusnady G. Fasman G. Protein Eng. 1991; 4: 669-679Crossref PubMed Scopus (329) Google Scholar, 30Perczel A. Park K. Fasman G.D. Anal. Biochem. 1992; 203: 83-93Crossref PubMed Scopus (421) Google Scholar). Wild-type pro-SPE B and mutants Q186N, C192S, H340R, N356D, and W357A without their signal peptides were expressed in an E. coli pET-21a expression system. All recombinant proteins contained 11 extra vector residues (ASMTGGQQMGS) and 6 histidine residues to simplify the purification procedures. All recombinant proteins were purified to apparent homogeneity in a single step by Ni2+ chelating chromatography. Recombinant wild-type streptopain was expressed as 42-kDa pro-SPE B and converted into a 28-kDa active enzyme during the course of purification. To prepare 42-kDa pro-SPE B, 1 mm HgCl2 was added as an inhibitor to prevent the conversion. In contrast, all mutant proteins were purified as 42-kDa zymogens without adding HgCl2 as an inhibitor. However, they formed inclusion bodies when the cells were induced at 37 °C. To obtain soluble protein and to maximize the yield, the cells were grown at different temperatures and induction periods. We determined the relative proportions of overexpressed proteins in the soluble and insoluble fractions that were obtained by varying the induction conditions. We found that 100, 68, 42, and 57 of the C192S mutant protein were soluble when the C192S mutant cells were induced at 28, 30, 32, and 37 °C, respectively. Similar approaches were used to obtain 1007 soluble proteins for other mutants. The optimal induction temperatures for the cells of H340R, N356D, and W357A mutants were 28, 25, and 17 °C, respectively. The final yields of the purified wild-type and Q186N, C192S, H340R, N356D, and W357A mutant proteins were ∼45, 40, 380, 210, 15, and 8 mg/liter, respectively (Table I). Based on SDS-PAGE analysis, the wild-type and mutant proteins were homogeneous (Fig. 2). Wild-type streptopain was freshly prepared to prevent autodegradation. The mutant proteins remained stable as 42-kDa zymogens at −20 °C for >2 years. The experimental molecular mass of the C192S mutant was determined to 42,334 Da with a deviation of <0.5 compared with the calculated value.Table IExpression and purification of wild-type streptopain and its Q186N, C192S, H340R, N356D, and W357A mutantsClonesProtein expression conditionsYield1-aYield indicates total soluble protein after purification by Ni2+ chelating chromatography.Specific activity1-bOne enzyme unit is defined as the amount of soluble protease required to release 1 ॖg of soluble azopeptides/min. ND, not detectable.mg/literunits/mgWild-type37 °C, 24 h45652 ± 52Q186N37 °C, 15–17 h40366 ± 30C192S28 °C, 12–14 h380NDH340R28 °C, 12–14 h210NDN356D25 °C, 12–14 h151.02 ± 0.2W357A15 °C, 12–14 h or 37 °C, 2 h1-cRefolding was required (protocol described under “Experimental Procedures”).80.013 ± 0.0051-a Yield indicates total soluble protein after purification by Ni2+ chelating chromatography.1-b One enzyme unit is defined as the amount of soluble protease required to release 1 ॖg of soluble azopeptides/min. ND, not detectable.1-c Refolding was required (protocol described under “Experimental Procedures”). Open table in a new tab To examine the structural differences between native and recombinant streptopain while excluding mutation effects due to conformational changes, we performed CD analyses to determine the secondary structures of wild-type and mutant streptopain. The CD spectra were fit with a root mean square deviation of 2–57 using the convex constraint algorithm together with the least-square fitting program (LINCOMB) of Perczel et al. (29Perczel A. Hollosi M. Tusnady G. Fasman G. Protein Eng. 1991; 4: 669-679Crossref PubMed Scopus (329) Google Scholar, 30Perczel A. Park K. Fasman G.D. Anal. Biochem. 1992; 203: 83-93Crossref PubMed Scopus (421) Google Scholar). Although a slight difference at ∼210 nm was observed, CD analysis showed that recombinant and native 28-kDa streptopain had 18.8 and 20.27 α-helix, 55.5 and 52.57 औ-structure, and 25.7 and 27.37 coil, respectively (Fig. 3A). These values are consistent with the reported x-ray structure of the pro-SPE B C192S mutant, which can be calculated by ignoring the propeptide region (26Kagawa T.F. Cooney J.C. Baker H.M. McSweeney S. Liu M. Gubba S. Musser J.M. Baker E.N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2235-2240Crossref PubMed Scopus (93) Google Scholar). In addition, the CD spectra of the 42-kDa zymogen forms of Q186N, C192S, H340R, N356D, and W357A were similar (Fig.3B). CD analysis showed that they contained 19.1–23.57 α-helix, 51.5–54.27 औ-structure, and 22.2–25.27 coil, respectively. Their secondary structures obtained from CD spectra were similar to the reported three-dimensional structure of the pro-SPE B C192S mutant, which contains 23.67 α-helix, 52.57 औ-structure, and 23.97 coil (26Kagawa T.F. Cooney J.C. Baker H.M. McSweeney S. Liu M. Gubba S. Musser J.M. Baker E.N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2235-2240Crossref PubMed Scopus (93) Google Scholar). We used azocasein as the substrate to examine the proteolytic activities of the wild-type and mutant proteins of streptopain. As shown in Table I, mutants C192S a" @default.
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• W1968839949 date "2003-05-01" @default.
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• W1968839949 title "Maturation Processing and Characterization of Streptopain" @default.
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