Keratinocyte Growth Factor-1(KGF-1) Expression In Insect Cells And Bioactivity Analysis

Keratinocyte growth factor1(KGF-1, FGF7) is the most effective and specific member of FGF family for skin damage repairing on epithelial cells. KGF-1, derived from mesenchymal cells, play an important role in the epithelial cells proliferation and differentiation of different organs through paracrine mainly dependent on its special receptor of FGFR2Ⅲb. KGF-1functions involved in epithelial morphology, wound epidermal cells regeneration, hair follicle growth, early lung morphogenesis and other physiological functions. The commercial pharmaceutical of KGF-1is Kepivance(?)(palifermin) that from Amgen used for oral mucositis caused by radiotherapy and chemotherapy. Furthermore, there are still some potential applications for chronic wound healing caused by diabetes and other metabolic diseases, a major obstacle in preterm infants who lacking of surfactant and bronchopulmonary dysplasia, lung injury and acute liver injury and regeneration. However, due to the low production of rKGF-1protein expression (E. coli) and instability, the development of KGF-1in pharmaceuticals was limited. KGF-1was also attempted to express in mammalian cells, plant cells, but the yield level and instability is still not resolved. So we try to express rhKGF-1in insect cells, because insect cell-baculovirus expression system (IC-BEVS) has been widely used in the last30years. There have been commercial vaccines in the market and thousands of foreign recombinant proteins yield by this expression system. The advantages of this system involved that it could be fast and efficient expressed even in a relatively large target protein, not containing animal pathogens, and the protein from eukaryotic could be modified with the most similar activity to the natural protein. Therefore, the feasibility of insect cells as bioreactor to produce recombinant keratinocyte growth factor-1was studied, accordingly to the problems of KGF-1protein to solve by following these solutions:Firstly, we improved expression of KGF-1from the genetic level. After the KGF-1gene mutant by insects preferred codon usage patterns, taking into account of the structure of mRNA and enhancement the stability; Furthermore, KGF-1is fairly unstable, so the gene of N-terminal amino acid was truncated based on existing reports, and three forms reconstructed baculovirus vectors; Secondly, there exists characteristics of post-translational modification in insect cell reactor, rhKGF-1protein retaining the natural signal peptide sequence, functions can be analyzed in insect cells; Thirdly, the IC-BEVS system is a transient expression system, cells lysate during the stage of infection phase, so the protein expression conditions and harvest time are directly impact the quality and yield of recombinant protein; As one of KGF-1characteristic is the heparin binding, so the rhKGF-1protein purification dependent on that structure; Finally, rhKGF-1biological activity in vitro was tested by MTT methods to assay BaF3and HaCat cells proliferation, in vivo activity assayed by preventing liver from acute injury and comparison effect of different KGF-1. Five results were obtained in the fowling:Firstly, optimized gene of KGF-1by insect preference codon can be expressed in insect cells (namely rhKGF-1194, rhKGF-1163, rhKGF-1140), and expression methods is different from three forms KGF-1proteins; native signal peptide of KGF-1played a key role in secretary expression in insect cells; Furthermore, different MOIs transfected cells have a significant effect on cell toxicity and protein yield, and the harvesting time is significantly different with the yield of protein, intracellular expression treated with MOI of1～3in84-96h, we got the best harvest, and the secretion is earlier in48-60h; Secondly, the stability of three forms protein was different, rhKGF-1140is more stabile than rhKGF-1194and rhKGF-1163; rhKGF-1194and rhKGF-1163appeared degradation during purification and storage, and the size of them were decreased from more than20kDa to17kDa near rhKGF-1140; Finally, three molecular protein have significant increasing the HaCat cells and BaF3cells proliferation, and rhKGF-1140is better than rhKGF-1194and rhKGF-1163; In vivo, rhKGF-1prevents the carbon tetrachloride-induced acute liver injury in mice, and rhKGF140is more efficient than rhKGF-1194and rhKGF-1163, rhbFGF also have a protective effect on acute liver injury in mice, but not significant; rhKGF-1could protect HL7702cells from the CCl4caused injury by decreasing necrosis and inhibiting apoptosis.In this study, the system of insect cell expression rhKGF-1was initially established and optimized, at the same time we also found the degradation of rhKGF-1during purification and storage. rhKGF-1expressed in insect cell reactor is feasible, the foundation by using insect cells was to produce rhKGF-1protein. But, it is still a long way to use insect cell reach to large scale production. Because the expression of conditions in this study was only optimized in small-scale by shaking flask, if using the wave or fermenter to enlarge the expression scale not only with MOI or TOI, but also need more conditions such as dissolved oxygen, pH, osmotic, medium nutrition and so on. It notable that the medium cost is very expensive. It is reported that serum-free medium made by their own formulation could greatly reduce the costs down10to20fold, so large-scale production of rhKGF-1from insect cell will take more time.