Bacterial cell surface display of foreign enzymes using autotransporter = Autotransporter를 이용한 외래 단백질의 박테리아 표면 발현

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The functional expression of foreign proteins on the surface of bacteria has proved important for numerous biotechnological applications. The outer membrane esterase (EstA) from Pseudomonas putida 3SK is a member of lipolytic autotransporter enzymes which possess an active site defined by the residues GDSL and contain the translocator domain promoting the translocation of the N-terminal passenger domain across gram-negative bacterial outer membranes. We investigated display of a foreign protein, the periplasmic enzyme β-lactamase (Bla), on the surface of Escherichia coli using the translocator domain of EstA. To find out the transport function of a C-terminal domain of EstA, we generated a set of the Bla-EstA fusion proteins containing N-terminally truncated derivatives of the EstA C-terminal domain. The surface exposure of the Bla moiety was verified by whole-cell immunoblots, protease accessibility and fluorescence-activated cell sorting (FACS). The investigation of growth kinetics and host cell viability showed that the presence of the EstA translocator domain in the outer membrane neither inhibits cell growth, nor affects cell viability. Furthermore, the surface-exposed Bla moiety was shown to be enzymatically active. These results demonstrate for the first time that the translocator domain of a lipolytic autotransporter enzyme is an effective anchoring motif for the functional display of foreign protein on the surface of E. coli. This investigation also provides a possible topological model of the EstA translocator domain, which might serve as a basis for the construction of fusion proteins containing foreign passengers. Due to the fact that EstA belongs to a family of lipolytic autotransporter enzymes, we suppose that the β-barrel formed by EstA might be appropriate for surface display of lipolytic enzymes, especially for lipases from related Pseudomonas species. Pseudomonas lipases play an important role in biotechnology both as hydrolases for detergent ...
Advisors
Seo, Yeon-Sooresearcher서연수researcher
Description
한국과학기술원 : 생명과학과,
Publisher
한국과학기술원
Issue Date
2004
Identifier
240580/325007  / 000995221
Language
eng
Description

학위논문(박사) - 한국과학기술원 : 생명과학과, 2004, [ ix, 131 p. ]

Keywords

FACS; GDSL; EstA; 유전자; 세포; 박테리아

URI
http://hdl.handle.net/10203/27575
Link
http://library.kaist.ac.kr/search/detail/view.do?bibCtrlNo=240580&flag=dissertation
Appears in Collection
BS-Theses_Ph.D.(박사논문)
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