Expression of streptomyces griseus trypsin gene in recombinant streptomycete and its purification = 재조합 스트렙토마이세스 균주에서 스트렙토 마이세스 그리세우스 트립신의 발현 및 분리 정제

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Streptomyces griseus trypsin (SGT) is one of the extracellular proteinases, which is produced by S. griseus. Low concentration of SGT in Pronase which was obtained from the culture broth of S. griseus made it difficult to purify SGT in pure form. Therefore the sprT gene which encodes premmature SGT protein was cloned in the plasmid pWHM3, a Streptomyces - E. coli shuttle vector. When the recombinant plasmid was introduced in S. lividans TK24, two proteins with molecular weight of 28 kDa and 42kDa respectively were detedted. The protein with a molecular weight of 28 kDa was a SGT protein and larger protein with a size of 42 kDa was supposed to be a premature form of the SGT protein. The SGT protein was purified to homogeneity in via of many steps of chromatographies, such as CM-sepharose chromatography, Mono-S chromatography and Superose-12 chromatography from the culture of S. lividans TK24 harboring the sprT gene. The SGT proteins from the Pronase and the transformant were compared for their N-terminal amino acid sequence, isoelectric point optimal pH and optimal temperature. All the properties studied above were the same in both SGT. Those results suggested that the sprT gene from S. griseus was successfully expressed in the heterologous host, s. lividans TK24 and the amount of the expressed SGT was reached to 5 times at least when it was calculated by the enzymatic activity against artificial substrate. This approach will be very helpful for the study fo Streptomycete proteases to elucidate its regulatory and processing mechanisms and its three-dimensional structure.
Advisors
Chung, Jae-HunresearcherByun, Si-Myungresearcher정재훈researcher변시명researcher
Description
한국과학기술원 : 생물과학과,
Publisher
한국과학기술원
Issue Date
1995
Identifier
101810/325007 / 000933024
Language
eng
Description

학위논문(석사) - 한국과학기술원 : 생물과학과, 1995.8, [ vi, 58 p. ]

URI
http://hdl.handle.net/10203/28477
Link
http://library.kaist.ac.kr/search/detail/view.do?bibCtrlNo=101810&flag=dissertation
Appears in Collection
BS-Theses_Master(석사논문)
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