Internal signal sequence within esterase I of pseudomonas fluorescens = Pseudomonas fluoressens의 esterase I에 존재하는 Internal signal sequence

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Unlike other secretory proteins, Esterase I of Pseudomonas fluorescens cloned into E. coli is secreted to the periplasmic space without typical hydrophobic $NH_2$-terminal signal sequence. It contains an internal hydrophobic α-helical structure ($^178QTLQIALLASLKA^190$) deduced by computer using the method of Chou and Fasman and it was supposed to function as internal signal sequence. To confirm this region is really responsible for the translocation of the enzyme, 5 substitution mutations and 1 deletion mutation (184 to 190) were performed on this region. All the mutations reduced the translocation of esterase I. When Leu188 was replaced to Asp, Leu184 to Glu, Leu185 to Pro, respectively, and residues 184 to 190 were deleted, the translocation was completely inhibited. When Ile182 was replaced to Asn, and Leu180 to Arg, respectively, secretion of Asn mutant (7%) and Arg, mutant (15%) was also reduced compared to wild-type (34%). The mutations also affected the activity of esterase. The activity of Asp, Glu, Pro and Deletion mutant was almost completely reduced, but the activity of Asn and Arg was about same that of wild-type. The stability of the enzyme was also affected by mutation. considering the thickness of esterase band of western blot and peak of esterase in densitometric scanning, the Asp, Glu, and Deletion mutant enzyme was less stable than wild-type enzyme. These results indicate that the internal hydrophobic α-helical region play an important role in translocation of Esterase I from cytoplasm to periplasm and that this region might be involved in catalytic activity of the enzyme, though the possibility of conformational change of the enzyme can not be ruled out.
Advisors
Yoo, Ook-Joonresearcher유욱준researcher
Description
한국과학기술원 : 생물공학과,
Publisher
한국과학기술원
Issue Date
1992
Identifier
60371/325007 / 000901454
Language
eng
Description

학위논문(석사) - 한국과학기술원 : 생물공학과, 1992.2, [ vii, 58 p. ]

URI
http://hdl.handle.net/10203/27755
Link
http://library.kaist.ac.kr/search/detail/view.do?bibCtrlNo=60371&flag=dissertation
Appears in Collection
BS-Theses_Master(석사논문)
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