Journal of Nippon Medical School: Vol.68 No.3 page.222

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ArticleTitle Cloning of Group A Streptococcal Pyrogenic Exotoxin-B Gene and its Recombinant Protein Expression in Culture Supernatant

AuthorList Yukino Watanabe

Affiliation

Language JA

Volume 68

Issue 3

Year 2001

Page 222-232

Received December 4, 2000

Accepted December 19, 2000

Keywords group A streptococcus, streptococcal pyrogenic exotoxin B, cysteine protease, molecular cloning, secreted recombinant protein

Abstract Streptococcal pyrogenic exotoxin B, a conserved cysteine protease (SPE B/SCP) released by group A Streptococcus (GAS) strains, is considered to be an important virulence factor of this bacterium. This paper reports the cloning of gene encoding SPE B/SCP. For production of recombinant SPE B/SCP (rSPE B/SCP), the primers specific for the SPE B/SCP gene (spe b) were designed based on its nucleotide sequence. Polymerase chain reaction (PCR) was performed with the genomic DNA of GAS strain NZ131 as a template. The amplified PCR products were purified and cloned into the pBluescript II SK⁺ plasmid vector. The vector was transformed into Escherichia coli (E. coli) JM109. The rSPE B/SCP and its recombinant proenzyme (rzym) were secreted in the culture supernate of the transformant. The rSPE B/SCP was purified from the supernatant by sequential chromatography on DEAE-Sepharose, matrix gel Red A and Sephadex G-50 columns. The purified rzym and rSPE B/SCP, respectively, gave a single band with a molecular weight approximately 40 kDa and 27 kDa on SDS-polyacrylamide gel electrophoresis, and reacted with anti-SPE B/SCP antibodies in Western Blot analysis. This is the first report in which rSPE B/SCP was obtained from the culture supernate of the transformant.

Correspondence to Yukino Watanabe, Department of Immunology and Infectious Diseases, Institute of Gerontology, Nippon Medical School, 1-396 Kosugi-cho, Nakahara-ku, Kawasaki, Kanagawa 211-8533, Japan
wtnbykn-nms@umin.ac.jp

ArticleTitle	Cloning of Group A Streptococcal Pyrogenic Exotoxin-B Gene and its Recombinant Protein Expression in Culture Supernatant
AuthorList	Yukino Watanabe
Affiliation
Language	JA
Volume	68
Issue	3
Year	2001
Page	222-232
Received	December 4, 2000
Accepted	December 19, 2000
Keywords	group A streptococcus, streptococcal pyrogenic exotoxin B, cysteine protease, molecular cloning, secreted recombinant protein
Abstract	Streptococcal pyrogenic exotoxin B, a conserved cysteine protease (SPE B/SCP) released by group A Streptococcus (GAS) strains, is considered to be an important virulence factor of this bacterium. This paper reports the cloning of gene encoding SPE B/SCP. For production of recombinant SPE B/SCP (rSPE B/SCP), the primers specific for the SPE B/SCP gene (spe b) were designed based on its nucleotide sequence. Polymerase chain reaction (PCR) was performed with the genomic DNA of GAS strain NZ131 as a template. The amplified PCR products were purified and cloned into the pBluescript II SK⁺ plasmid vector. The vector was transformed into Escherichia coli (E. coli) JM109. The rSPE B/SCP and its recombinant proenzyme (rzym) were secreted in the culture supernate of the transformant. The rSPE B/SCP was purified from the supernatant by sequential chromatography on DEAE-Sepharose, matrix gel Red A and Sephadex G-50 columns. The purified rzym and rSPE B/SCP, respectively, gave a single band with a molecular weight approximately 40 kDa and 27 kDa on SDS-polyacrylamide gel electrophoresis, and reacted with anti-SPE B/SCP antibodies in Western Blot analysis. This is the first report in which rSPE B/SCP was obtained from the culture supernate of the transformant.
Correspondence to	Yukino Watanabe, Department of Immunology and Infectious Diseases, Institute of Gerontology, Nippon Medical School, 1-396 Kosugi-cho, Nakahara-ku, Kawasaki, Kanagawa 211-8533, Japan wtnbykn-nms@umin.ac.jp