| Epidermal Growth Factor(EGF)is a polypeptide composed of 53 amino acids with a molecular weight of 6054 Da.EGF has a wide range of functions,which can not only promote the proliferation and differentiation of epidermal cells,but also promote the growth and migration of keratinocytes,thus promoting the proliferation of fibroblasts and embryonic cells.Therefore,EGF plays an important role in wound healing,accelerating wound repair and organ formation.It is also widely used in various cosmetics because it can promote the synthesis and secretion of hyaluronic acid,glycoprotein,etc.,and activate the basal cells of skin.With the deepening of research on EGF,its special biological effects have been continuously explored and become the research focus in recent years.Pichia pastoris expression system is the most widely used expression system in recent years.Expression vectors in this system are all integration vectors,among which pGAP series has more prominent advantages than other integration vectors.First of all,methanol does not need to be added during fermentation using pGAP system.On the one hand,it can save the transportation cost of methanol,on the other hand,it can also avoid the potential danger of methanol due to flammability and explosion.Secondly,the carbon source does not need to be replaced in the fermentation process,thus reducing the pollution of miscellaneous bacteria.In addition,the pGAP expression system adopts a continuous fermentation mode,the fermentation process is simple and the period is short,thus greatly improving the production efficiency and being more beneficial to industrial production.The purpose of this study is to express EGF constitutively using Pichia pastoris expression system of glyceraldehyde triphosphate dehydrogenase promoter(pGAP)and to optimize the fermentation process.In this study,the gene sequence of EGF was optimized and synthesized based on Pichia pastoris expression vector PGAZ? A according to codon preference of yeast cells.Xhol I and Eco R I sites were introduced at both ends of the sequence.In order to facilitate purification,6×HIS was introduced before the target gene.In order to realize efficient protein expression,fusion tag-Asn-Gly was selected to construct recombinant plasmid PGAZ? A-6HIS-Asn-Gly-EGF.Aftersequencing is correct,linearized electricity is transferred to Pichia pastoris competent state GS115.Engineering bacteria successfully integrated into yeast genome were screened by Zeocin resistance medium and PCR identification.Engineering bacteria with high expression of recombinant protein were obtained by shake flask culture and Tricine-SDS-PAGE analysis.The study also explored the fermentation process and optimized the fermentation conditions by single factor experiment method: the best carbon source,the best pH value,the best carbon source concentration,the best fermentation temperature,fermentation time and inoculation concentration.The recombinant protein was purified by Ni-Sepharose 6FF affinity chromatography column and EGF secretion concentration was detected by Bradford method.Finally,the proliferation activity of recombinant protein cleaved by hydroxylamine on Balb/c3T3 cells was determined.Through experiments,it was found that the highly expressed strain had the highest protein expression of 0.05mg/mL at 72 h.After optimization of fermentation conditions,the optimal carbon source is glycerol,its concentration is 2.5%,fermentation time is72 h,inoculum size is 2.0%,pH is 6.0,and temperature is 30?.Purified recombinant protein was successfully obtained from supernatant of fermentation broth by Ni-Sepharose 6FF column.Biological activity test results show that the recombinant protein can effectively promote the proliferation of Balb/c 3T3 cells in a dose-dependent manner after being cut by hydroxylamine,and has good biological activity. |
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