Anchorage of cyclodextrin glucanotransferase on the outer membrane of Escherichia coli

Abstract

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.

Development of an Autofluorescent Whole-Cell Biocatalyst by Displaying Dual Functional Moieties on Escherichia coli Cell Surfaces and Construction of a Coculture with Organophosphate-Mineralizing Activity

“…The LppOmpA and INP systems used in this study look promising, since they are well suited as a carrier of relatively large inserts. The largest proteins that have been successfully displayed with Lpp-OmpA and INP in E. coli so far are a 74-kDa cyclodextrin glucanotransferase and a 90-kDa chitinase, respectively (42,45).…”

“…The Lpp-OmpA-based cell display system has been extensively used for the display of heterologous proteins, such as ␤-lactamase (11), cellulases (12), the scFv antibody (6), cyclodextrin glucanotransferase (42), and chitinbinding domain (43), on the surface of E. coli.…”

Exploring Surface Display Technology for Enhancement of Delivering Viable Lactic Acid Bacteria to Gastrointestinal Tract

“…The mislocalization of target proteins can affect their functions negatively ( Dieye et al, 2003;Van Der Vaart et al, 1997;Wan et al, 2002). In contrast, the surface display systems based on the external mode of protein display can ensure the full exposure of target proteins outside of the cell wall and the surface intensity of target proteins can readily be adjusted by selecting appropriate display hosts and suitable concentrations for the fusion proteins in the incubation mixture.…”

Novel Bacterial Surface Display Systems Based on Outer Membrane Anchoring Elements from the Marine Bacterium Vibrio anguillarum

“…Lpp-OmpA is an efficient surface display system developed by Georgiou et al (9), which has been used successfully to anchor a variety of proteins, including some enzymes, onto the cell surface (10,21). This display system allows C-terminal fusion of the passenger proteins and consists of two key anchoring motifs: (i) the signal sequence and the first nine amino acids of E. coli lipoprotein (Lpp), to target the proteins to the inner face of the outer membrane, and (ii) the transmembrane region (amino acids 46 to 159) of E. coli outer membrane protein A (OmpA), to conduct the proteins across the outer membrane.…”

“…This display system allows C-terminal fusion of the passenger proteins and consists of two key anchoring motifs: (i) the signal sequence and the first nine amino acids of E. coli lipoprotein (Lpp), to target the proteins to the inner face of the outer membrane, and (ii) the transmembrane region (amino acids 46 to 159) of E. coli outer membrane protein A (OmpA), to conduct the proteins across the outer membrane. Since the Lpp-OmpA mode combines the anchoring capabilities of lipoprotein and outer membrane protein, it has outstanding advantages in high surface display efficiency and strong adaptability to passenger proteins varying in size (21).…”