The Construction Of An Secretory Expression Vector Based On PHY300PLK And The Expression Of The Exogenous Gene

Shuttle vector pHY300PLK is frequently used as the backbone for the construction of expressing vectors. The expression system of heat-stable a-amylase (amy) gene of Bacillus Hcheniformis including the promoter, signal peptide and amylase gene was cloned into pHY300-PLK.The anti-Ampcillin gene of the recombinant plasmid was deleted by inverse PCR and homologous recombination for efficient expression of endogenous-proteins and exogenous proteins using the promoter and signal peptide of a-amy gene. The result indicated that the pHY300PLK with a-amylase gene, namely, plasmid pAMY, could express a-amylase in host strain while the plasmid plasmid pAMYl could express the a-amylase more efficiently. According to compare the growth curve of recombinant strains and the plasmid copy number of pAMY and pAMYl, deletion of the anti-Ampcillin showed no influence on the growth of the recombinant strain and improved the replication efficiency of the plasmid, leading to higher expression of protein by pAMYl.Then the cellulase DL-3gene was inserted into pAMYl after the promoter and signal peptide of a-amy gene.This recombinant plasmid, named pCEL, could express cellulase in host strain. There was a55kD protein band by SDS-PAGE of cell free supernatant of the culture, which was consistent with the cellulose DL-3molecular weight. And the cellulase activity was45U/mL.These results indicated the promoter and signal peptide of a-amy gene could promote not only a-amylase but also the exogenous gene as well.Moreover,the gene encoding proteinase K was cloned into expressing vector pAMYl, resulting in recombinant plasmid pPRO,The proteinase K was expressed successfully in this recombinant vector, which could hydrolyze milk protein, indicating the successful expression of another exogenous gene besides cellulase gene using pAMYl.The enzymatic activity of the purified proteinase K under pH8.0at37癈is1000U/mL.The activity of proteinase K is optimal when the pH is10.0,which is too high to food industry. But this enzyme still exhibited55%activity under pH6-9or pH11-12.The above results suggested that the cloning vector pHY300PLK was successfully modified to expressing vector and could be used to express exogenous genes.