High level expression of bacillus subtilis endo-β-1,4-glucanase gene in escherichia coliBacillus subtilis 섬유소 분해효소의 Escherichia coli에서의 대량생산

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A Bacillus subtilis endo-β-1,4-glucanse gene, previously cloned in our laboratory, was subcloned in expression vectors, pPLc2833 and pKK223-3. Plasmid pPLc2833 contains lambda $P_L$ promoter followed by multiple cloning site. Plasmid pKK223-3 contains the hybrid promoter tac which has the -35 region of trp promoter and -10 and operator region of lac operon followed by multiple cloning sites originated by pUC8. The endoglucanase gene-containing plasmid, pUBS102(8), was digested with appopriate restriction enzymes, and ligated with the restriction enzymetreated vectors. Those ligation mixtures were used to transform E. coli strains of M5248 and JM103. From the transformants, recombinant plasmids were isolated and named pLBS1 and pKBS1. In pLBS1 the structural gene of the endoglucanase was inserted under the control of promoter $P_L$ from pPLc2833, whereas in pKBS1 the same structural gene was inserted under the control of promoter Ptac from pKK223-3. E. coli M5248 was transformed with pLBS1 and JM103 was with pKBS1. E. coli M5248(pLBS1) was grown in LB medium at 28℃ until the O.D.600 value reached 0.4-0.5. When the culture temperature was shifted to 42℃ the endoglucanase production was induced to reach up to about 1.5 times of that of uninduced cells in 3 h, and followed by a plateau. When the culture of E. coli JM103 (pKBS1) was induced with IPTG, the enzyme production reached 15 times higher than that of the uninduced cells. The actual production of the endoglucanase protein was much higher than the enzyme activity and reached 23% of the total cell protein suggesting that the new protein produced was remained in E. coli cells in an inactive form.
Advisors
Pack, Moo-Young박무영
Description
한국과학기술원 : 생물공학과,
Publisher
한국과학기술원
Issue Date
1989
Identifier
66656/325007 / 000871318
Language
eng
Description

학위논문(석사) - 한국과학기술원 : 생물공학과, 1989.2, [ v, 54 p. ]

URI
http://hdl.handle.net/10203/28319
Link
http://library.kaist.ac.kr/search/detail/view.do?bibCtrlNo=66656&flag=dissertation
Appears in Collection
BS-Theses_Master(석사논문)
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