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KMID : 0545119980080040333
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Journal of Microbiology and Biotechnology
1998 Volume.8 No. 4 p.333 ~ p.340
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Purification and Characterization of Streptomyces griseus Trypsin Overexpressed in Streptomyces lividans
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Koo, Bon Joon
Bae, Kwang Hee/Byun, Si Myong/Hong, Soon Kwang
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Abstract
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Streptomyces griseus trypsin (SGT) is an extracellular proteinase produced by S. griseus. The sprT gene, which encodes premature SGT protein, was cloned into the plasmid pWHM3, a Streptomyces-E. coli shuttle vector. When the recombinant plasmid was introduced into Streptomyces lividans TK24, two proteins with molecular weights of 28 kDa and 42 kDa were detected. The 28-kDa protein was a SGT protein while the larger 42-kDa protein is thought to have been a premature form of the SGT protein. The SGT protein was purified to homogeneity via ammonium sulfate fractionation and many column chromatographies, including CM-sepharose chromatography, Mono-S chromatography, and Superose-12 chromatography, from the culture broth of S. lividans TK24 harboring the sprT gene. The N-terminal amino acid sequence, isoelectric points, and stabilities at various conditions of the SGT proteins purified from the Pronase and transformant were almost identical. The amount of the expressed SGT in S. lividans TK24 was determined to be 5 times more than that of S. griseus based on the enzymatic activity against artificial substrate.
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