The Expression of Recombinant Human Hepatocyte Growth Factor in Pichia pastoris

Abstract

The aim of this study was to construct a stable and efficient eukaryotic expression system for the secretion of biologically active recombinant human hepatocyte growth factor (rhHGF). The eukaryotic expression vector pGAPZα A was chosen to express rhHGF. To ensure the presence of the secondary structure, we inserted the enterokinase sequence between Arg494 and Val495. After digesting the rhHGF and pGAPZα A plasmid with Xho I and Xba I, we connected and transformed them into E. coli Trans10 competent cells. This resulted in the successful construction of the shuttle plasmid, pGAPZα A-rhHGF. After sequencing, we transformed the linearized pGAPZα A-rhHGF plasmid into Pichia pastoris GS115 using electroporation for subsequent protein expression. The expressed rhHGF samples were collected at 0, 24, 48, 72 and 96 h, purified by affinity chromatography, and tested using Western blotting. As a result, the pGAPZα A-rhHGF shuttle plasmid was constructed successfully. A positive band of approximately 80 kDa was observed in the Western blotting indicating successful expression of rhHGF. The highest expression abundance of rhHGF protein was observed at 48 h. Furthermore, we isolated and cultured primary rat hepatocytes, the harvested rhHGF protein exhibited high biological activity. This research provides experimental evidence for the eukaryotic expression of rhHGF protein and theoretical support for large-scale manufacturing.