Application of over lap PCR to construction of expression vector for sodAP gene

Objective: To construct an recombinant plasmid for expression of sodAP gene of Arthrobacter pascens DMDC12 in E. coli BL21 (DE3) by over lap PCR. Methods: Promoter P121 and sodAP gene were amplified using two pairs of designed primers respectively, then linked by over lap PCR, identified by sequencing and inserted into prokaryotic expression vector pET-22b (+). Transform the constructed recombinant plasmid pET-PsodAP to E. coli BL21 (DE3) and identify the expressed product by SDS-PAGE. Determine the expression level of target protein by quantitative photodensitometry. Results: The linked P121 promoter and sodAP gene showed a band of about 800 bp on gel electrophoretic profile, and its sequence was consistent with that respected. Restriction analysis proved that recombinant plasmid pET-PsodAP was constructed correctly. The expressed product contained 19% of total somatic protein. Conclusion: The recombinant expression vector for sodAP gene was successfully constructed by over lap PCR technique and expressed in E. coli BL21 (DE3), which laid a foundation of industrial production of Mn-SOD.