Expression Of Recombinant TAT-human Epidermal Growth Factor In Pichia Pastoris

Epidermal Growth Factor(EGF)is a kind of small molecular polypeptide containing 53 amino acids,which has a stable structure and the molecular weight is about 6.0k Da.EGF can promote cell proliferation,differentiation and migration,and participate in embryonic development,tissue regeneration and other physiological processes.It is used in the clinical treatment of all kinds of trauma,ulcer and corneal injury,such as burn and scald.In addition,EGF can also accelerate the metabolism of normal epidermal cells.As cosmetic additives,it can play a role in delaying skin aging,whitening and skin rejuvenation.In recent years,the research on EGF,whether it is used as a medical or cosmetic additive,has great application value and market prospect which can not be underestimated.Cell-penetrating peptides(CPPs)is a polypeptide found in recent years that can effectively traverse the cell membrane,TAT protein belongs to the cationic cell transmembrane peptide,which is the trans acting factor of HIV-1,Studies have shown that TAT protein can mediate proteins and nucleic acids and other substances safely and effectively into the cell under the condition of not destroying the cell membrane,and the transduction is 47-57 of the 11 amino acid residues.As a new drug delivery tool,TAT protein has a unique advantage and has a wide application prospect.In this paper,the human epidermal growth factor(h EGF)and cell penetrating peptide TAT(47-57)is intended to improve the penetration ability of h EGF,make it easier to increase the penetration rate,so as to exert biological activity.The sequence of TAT(47-57)and h EGF amino acid sequence were connected in series.According to Pichia pastoris preference codon,TAT and h EGF gene sequence was designed without changing the amino acid sequence.Construction of eukaryotic expression vector of p PICZ?A-TAT-h EGF by gene recombination technique,after linearization power to Pichia pastoris X-33 by screening,identification,select the successful transformation of recombinant was induced by methanol,selected high expression strains,and the sixth day has the highest protein expression,it can reach about 0.6mg/m L,and the best induction p H value is 4.6.Shake flask expression induced by supernatant protein by isoelectric point precipitation,40% saturated ammonium sulfate salting out preliminary separation of protein concentration by gel filtration chromatography,using 30mmol/L acetic acid sodium acetate buffer(p H5.2)successfully eluted protein separation,then by ion exchange chromatography with 30mmol/L acetic acid sodium acetate buffer(p H5.2)300mmol/L Na Cl successfully purified fusion protein TAT-h EGF.The results of bioactivity test showed that the purified fusion protein could promote the proliferation of NIH/3T3.