High-level Expression,Purification And Bioactivity Assay Of Recombinant Human Keratinocyte Growth Factor-2 Fusion Protein

Ojective: To increase the expression level of human keratinocyte growth factor-2(hKGF-2) by fusing with small ubiquitin-related modifier (SUMO), and to obtain the purified hKGF-2 with biological activity.Method: The fusion DNA fragment composed of SUMO gene and hKGF-2 gene was amplified by PCR and inserted into the expression vector pET28a to construct the recombinant plasmid, pET28a—SUMO—hKGF-2. The plasmid was transformed into E.coli RosettaTM2(DE3) and the fusion protein SUMO—hKGF-2 was expressed by IPTG induction. SUMO-hKGF-2 was purified by Ni-NTA affinity chromatography from the supernatant of the bacteria lysate. The fusion proteins were hydrolyzed by the SUMO proteases to get the protein hKGF-2. The mitogenic activities of SUMO—hKGF-2 and hKGF-2 were detected by MTT.Result: The recombinant expression strain RossetaTM2(DE3)/pET28a—SUMO—hKGF-2 was successfully constructed, and the expression condition was optimized. The recombinant fusion protein SUMO—hKGF-2 was expressed at 30℃for 6h, with the induction of IPTG at the final concentration of 0.6mM. The purity of SUMO—hKGF-2 protein purified by Ni-NTA resin was about 79.4%, and this product was desalted and then hydrolyzed by the SUMO proteases. The result of mitogenicity assay showed that the purified hKGF-2 could significantly promote the proliferation of normal rat kidney epithelial (NRK52E) cells, the fusion protein SUMO-hKGF-2 also have the mitogenic activity, which were lower than that of hKGF-2.Conclusion: The recombinant expression strain Rosseta^TM2(DE3)/pET28a—SUMO—hKGF-2 was successfully constructed, which could express fusion protein SUMO—hKGF-2 at high level. The recombinant hKGF-2 was obtained via purification and SUMO protease digestion.