Expression Of Recombinant Human Tissue Kallikrein Using Baculovirus-Insect Cell Expression System

The baculovirus-insect cell expression system has been extensively used to express recombinant proteins, with relatively high yields and authentic post-translational modifications of the expressed products. To express recombinant human tissue kallikrein(KLK1)using the baculovirus-insect cell expression system, the klk1 cDNA was amplified by high fidelity PCR from a recombinant eukaryotic expression vector pOV4k with suitable restriction sites and 6xHis tag introduced. After digestion with restriction enzymes BamHI and XhoI, the PCR product was subcloned into baculovirus transfer vector pFastBac1 and the resultant recombinant vector pFastBacl-KLK1 was introduced into DH10Bac E.coli containing the shuttle vector Bacmid. By site-specific transposition, the klk1 cDNA was integrated into Bacmid and the recombinant shuttle vector Bacmid-klk1was generated. By transfection of Sf9 cells with Bacmid-KLK1, the recombinant baculovirus was obtained. After infection of Sf9 cells with the recombinant virus for 3 passages, the recombinant enzyme was detected by indirect fluorescence assay and Western Blot with the expected molecular weight of 35kDa. Most of the recombinant protein was present in the cytoplasm. The successful expression of recombinant KLK1 laid a foundation for development of reagents for KLK1 identification and functional studies.