| Pfu DNA polymerase derived from the Archaea Pyrococcus furiosus, with two subunits (P45and P50) and molecular weight of nearly90kDa. According to its5’-3’polymerase activity and3’-5’exonuclease activity, Pfu DNA polymerase could correct the mutation during the amplification and thus exhibit superb fidelity. Ssold is a double strand DNA binding protein of Sulfolobus solfataricus. Linked with the DNA polymerase, it could improve the processivity of polymerase and maintain the enzymatic activity and thermostability of polymerase.In order to explore a new method to produce Pfu DNA polymerase, the present study was carried out to express fusion protein of Pfu DNA polymerase and Sso7d, which was named pfusso, in Pichia pastoris. The pfusso DNA polymerase gene was amplified and constructed into Pichia pastoris secretion expression vector pHBM905A, which contains the methanol oxidase promoter and drives the expression of heterologous proteins in Pichia pastoris precisely. After lineration, the constructed plasmid was transformed into GS115competent cell. The transformants were chosen randomly and induced by methanol at the concentration of0.5%. All these induced specimens were sampled per24hours and analyzed by SDS-PAGE. Besides, the supernatants of these specimens were utilized to perform the PCR as a polymerase. As is reviewed in the outcome, the secreted expression of pfusso DNA polymerase was accomplished in the Pichia pastoris. The total protein concentration in the medium supernatant was187mg/L, according to the data of Bradford Protein Concentration Assay. Moreover, the pfusso DNA polymerase expressed in the Pichia pastoris manifested higher amplification output and thermostability, yet lower fidelity, compared to the pfusso DNA polymerase expressed in E. coli.Therefore, this expression strategy conferred us a simple, efficient and economical approach to accomplish the production of Pfu DNA polymerase, laid a solid foundation for its industrial production. |
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