Expression Of Bacillus Subtilis WHNB02 Phtase PhyC Gene In E.coli And The Construction Of Mazie Expression Vector

The phytase phyC gene fragment from the Bacillus subtilis WHN02 (GenBank Accession No. AY220075) encoding mature enzyme was amplified by PCR with insertion of BamHI and SalI sites at the 5' and 3' ends, respectively .The amplified PCR fragment about l.lkb in length was purified with DNA purification kit. Purified phyC BamHIISalI was cloned to pUC18-T vetor After cut with BamHIISall ,phyC was ligated to E.coli expression vector pGEX-4T-l. Plasmid pGEX-phyC was transformed into the CaCl2-competent E.coli JM109 expression host. The positive transformants were grown in LB broth containing 100 g of ampicillin per ml, and induced by 0.1 mM isopropyl-p-D-thiogalactopyranoside(IPTG).The recombinant proteins from ultrasonic crash supernatant were assayed for phytase activity. SDS-PAGE showed that the molecular mass of recombinant protein was about 69kD.Full IPTG-dependent induction was achieved after 3h and protein was produced to a level as high as 47.4% of the total cellular protein.The phytase phyC gene fragment from the Bacillus subtilis WHN02 (GenBank Accession No. AY220075) encoding the phytase was amplified by PCR with insertion of BamHI and SacI sites at the 5' and 3' ends,respectively .The amplified PCR fragment about l.lkb in length was purified with DNA purification kit. Purified phyC BamHI/SacI was cloned to pUCm-T vetor After cut with BamHI/SacI ,phyC frgment was ligated to vector pBPC47,which contains maize specific albumen EGH5 promoter and NOS terminator .The recombinant transitional vector pBPC-phyC was constructed successfully. In terms of analyzing the vector pBPC47, pBPC-phyC was cut with BgIII/HindIII/Xhol. Cut fragment(EGH5-phyC-NOS) about 2.3kb in length was purified with DNA purification kit. Purified EGH5-phyC-NOS BgIII/HindIII/Xhol was cloned to maize expression vector pCAMBIA1300.The recombinant expression vector pB-C-phyC was obtained after the transformants were grown in LB plate .Ultimately it has transformed into maize successfully.