Wang He, Yang Rui-jin, Hua Xiao, Zhao Wei, Zhang Wen-bin
In this work,we developed an efficient spore display system that a model protein β-galactosidase was anchored on the spore surface of Bacillus subtilis 168 based on the use of spore coat proteins.The PCR-amplifying cotB,cotC,cotG and cotX were ligated with pMD-19T and digested with XbaI and KpnI,and then subcloned into vector pJS700a previously digested with the same two restriction enzymes,finally resulted in the plasmids pJSB,pJSC,pJSG and pJSX.To construct the gene fusions,the bgaB from Bacillus stearothermophilus IAM11001 was cloned into the KpnI and EcoRI sites of plasmid pJSB,pJSC,pJS G and pJSX to generate generating the plasmids pJSBB,pJSCB,pJSGB and pJSXB,respectively After linearization with BglII restriction endonuclease,the four recombinant integrative plasmids were transformed into B.subtilis 168 to yield the recombinant strain PB701,PB702,PB703 and PB704,respectively.Results from Western blot analysis showed that the fusion protein was immobilized on the spore surface.Using oNPG as substrate,the enzyme activity of spore-displaying β-galactosidase was assayed and they were 0.14,0.06,0.22 and 0.20 U/mL for PB701,PB702,PB703 and PB704,respectively.This result suggested that the fusion proteins could be successfully expressed and located on the spore surface of B.subtilis.
