Purification and Characterization of Streptomyces griseus Trypsin Overexpressed in Streptomyces lividans

  • KOO, BON-JOON (R&D Center, TS Corporation) ;
  • KWANG HEE BAE (Department of Biological Science, Korea Advanced Institute of Science and Technology (KAIST), and Research Center for New Bio-materials in Agriculture (RCNBMA)) ;
  • SI-MYONG BYUN (Department of Biological Science, Korea Advanced Institute of Science and Technology (KAIST), and Research Center for New Bio-materials in Agriculture (RCNBMA)) ;
  • SOON-KWANG HONG (Department of Biological Science, Myong Ji University)
  • 발행 : 1998.08.01

초록

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 TK 24 was determined to be 5 times more than that of S. griseus based on the enzymatic activity against artificial substrate.

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