Anchorage of cyclodextrin glucanotransferase on the outer membrane of Escherichia coli.
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% un-translocated. 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.
Department of Chemical Engineering, National Chung Hsing University, Taichung, 402, Taiwan.
Hsiao-Ming
HM
Wan
Ban-Yang
BY
Chang
Sung-Chyr
SC
Lin
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D005964:Glucosyltransferases / Q000096:biosynthesis* / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics
D001425:Bacterial Outer Membrane Proteins; D011993:Recombinant Fusion Proteins; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase