[Cloning of a phyA gene and its over expression in E. coli]

This research amplified the phyA gene with the designed and synthesized primers specific for the phyA gene full-length coding sequence. The phyA gene was from Aspergillus niger F246 by the polymerase chain reaction(PCR), which is selected and identified in our laboratory. After sequncing the coding sequence, it was confirmed that the construction of cloning vector was succeeded. The phyA gene fragment was recovered from the pMD18T-phyA and ligated with prokaryotic expression vector pET30a+ to construct the recombinant expression plasmid pET30a+ -phyA. It was expressed with IPTG induction in E. coli for high efficiency. A new protein band with apparent molecular weight 50 kDa was detected in the lysate of the transformed cell by using SDS-PAGE. The amount of the soluble fusion protein was about 40% of large intestine bacillus soluble protein of transformed cells, estimated by absorbance scanning of SDS-PAGE and protein quantitation. It's phytase activity was eight times over the natural phyase. So this research provides the basis of the study on obtaining large and high active phytase and developmant of the new microbial ecologicalagent.