Primary Construction Of A Novel Cell Surface Display System For The Chaperone-dependent Lipase

Various cell-surface display systems have been widely used as one kind of high-throughput screening technique in protein engineering. A large number of monomeric proteins have been succeededly displayed on the cell surface. However, the oligomeric proteins or the chaperone-dependent proteins were still difficult to be displayed because of the technological problem. In recently years, with the research progress on the crystal structure of autotransporters, the autotransporters-based surface display system for oligomeric proteins was occassionally reported. In this study, the transmembrane domain of EstA from pseudomonas aeruginosa was selected as the carrier protein to construct a novel Escherichia coli surface display system, which then used to functionally display lipase LipA from Burkholderia sp. ZYB002 and the corresponding lipase-specific foldase. The main results can be summarized as follows:(1). Investgation the localization of lipase-specific foldase (LipB) in E. coli on the display effect of lipase LipA.The fusion protein of EstA/LipA and the chaperone protein LipB was simultanelously expressed in E. coli while LipB was situated in the intracellular, periplasmic space and cell surface of E. coli, respectively. Unfortunately, regardless of the location of LipB in E. coli, no lipase activity on the E.coli cell surface was detected.(2). Investgation the length and the type of the linker between EstA and the foreign protein on the display effect.Bacillus subtilis lipase was fused with EstA at the position of Leu297, Ile292, Leu282 and Ser272, respectively. The highest whole-cell lipase activity was obtained when the lipase was fused with EstA at the position Leu297. On the contrary, eGFP couldn’t functionally displayed on E. coli cell surface with the same linker.(3). Investgating different autotransporters on display effect of different foreign protein.eGFP was fused to AG43 and EspP from E. coli, respectively. The whole cell fluorescence assay showed that eGFP was functionally displayed on the cell surface of E. coli.