Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/41130
標題: Anchorage of cyclodextrin glucanotransferase on the outer membrane of Escherichia coli
作者: Wan, H.M.
林松池
Chang, B.Y.
Lin, S.C.
關鍵字: cyclodextrin glucanotransferase;E. coli;surface display;ice-nucleation protein;cell-surface display;alkalophilic bacillus sp;pseudomonas-syringae;affinity maturation;bacterial-cells;libraries;expression;cellulase;peptides
Project: Biotechnology and Bioengineering
期刊/報告no:: Biotechnology and Bioengineering, Volume 79, Issue 4, Page(s) 457-464.
摘要: 
The gene encoding cyclodextrin glucanotransferase (CGTase) was successfully cloned from B. macerans by PCR. A recombinant plasmid pCS005 with a gene encoding the Lpp-OmpA-CGTase trifusion protein was constructed and transformed into E coli for the surface display of CGTase. Results of immunoblotting analysis and protease accessibility on the fractionated cell membranes confirmed that the Lpp-OmpA-CGTase trifusion protein was successfully anchored on the outer membrane of E coli. However, only 50% of the membrane-anchored trifusion proteins were displayed on the outer surface of E. coli with the remaining 50% untranslocated. The low efficiency of surface display is attributed to the large size of CGTase. Only a trace amount of CGTase activity was detected for both the whole cells and the cell debris fractions. Because the results of the protease accessibility study suggested that the trypsin-resistant conformation of CGTase was preserved in the membrane-anchored CGTase, we believe that the lack of enzyme activity is mainly due to the inaccessibility of the CGTase active site, near the N-terminus, for substrate molecules. It can be estimated that the critical size for surface display of protein in E. coli is approximately 70 kDa. (C) 2002 Wiley Periodicals, Inc.
URI: http://hdl.handle.net/11455/41130
ISSN: 0006-3592
DOI: 10.1002/bit.10301
Appears in Collections:化學工程學系所

Show full item record
 

Google ScholarTM

Check

Altmetric

Altmetric


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.