Development of systems for secretion and cell surface display of proteins in Escherichia coli

Abstract

Recombinant proteins can be synthesized in E. coli and guided to four different locations: the cytoplasm, the periplasmic space, the inner or outer membrane, and extracellular medium. Proteins found in the outer membrane or periplasmic space are synthesized in cytoplasm as premature (unprocessed) proteins. In E. coli, it has been established that protein secretion through the inner membrane to the periplasm or outer membrane is achieved by the signal peptide-dependent general secretion pathway (GSP) that is widespread as a universal secretion pathway. In this study, secretion and cell surface display system of proteins in E. coli for biotechnological application were developed. Firstly, new secretion vectors containing the Bacillus sp. endoxylanase signal sequence were constructed for the secretory production of recombinant proteins in E coli. The E. coli alkaline phosphatase structural gene fused to the endoxylanase signal sequence was expressed from the trc promoter in various E. coli strains by inducing with IPTG. Among the tested, E. coli HB101 showed the highest efficiency of secretion (up to 25.3% of total proteins). When cells were induced with 1 mM IPTG, most of the secreted alkaline phosphatase formed inclusion bodies in the periplasm. However, alkaline phosphatase could be produced as a soluble form without reduction of expression level by inducing with less (0.01 mM) IPTG, and greater than 90% of alkaline phosphatase could be recovered from the periplasm by simple osmotic shock method. Fed-batch cultures were carried out to examine the possibility of secretory protein production at high cell density. Up to 5.2 g/L of soluble alkaline phosphatase could be produced in the periplasm by the pH-stat fed-batch cultivation of E. coli HB101 harboring pTrcS1PhoA. New artificial sequence was designed by analysis of Gram-negative bacterial signal sequences. The secretion efficiency could be improved by mutagenic PCR of artificial signal sequence Secondly, a ...