Optimization Of Fermentation Of Yeast SMD1168H Expressing HGRNA

The GRN precursor is a secreted glycoprotein of about 88 kD in molecular weight,encoded by the hGRN gene.GRN is a secreted polypeptide with a molecular weight of 6 kD,which is formed by the decomposition of GRN precursors under the action of hydrolases.At present,researchers have isolated and purified a series of these peptides,such as hGRN A,B,C,D and F.Researchers found that hGRN A is abundant in epithelial cells and plays a crucial role in the nervous system,immunity and tumorigenesis.In this study,the DNA fragment with hGRN A was cloned as a template and two kinds of primer were constructed.Then a large number of DNA fragment were obtained by PCR,and then inserted into the plasmid pGAPZ?A by double enzyme digestion to obtain the recombinant expression vector----pGAPZ?A-hGRN A.To achieve the expression of the cloned hGRN A gene in eukaryotic system The obtained recombinant plasmids were linearized by single enzyme digestion,and then were transformed into yeast cells by electroporation and fused with the yeast genome.Response surface methodology was used to obtain the optimal yeast cell fermentation conditions to increase the expression of hGRN A.The high purity recombinant hGRN A was obtained by separation and purification of the fermentation product using MCAC column composed of Ni²⁺.The pharmacological activity of the recombinant hGRN A on tumor cells was determined by MTT assay.The effect of the recombinant hGRN A on the appearance of nuclear in tumor cells was observed using DAPI staining.The antitumor activity of recombinant hGRN A was further studied by FCM technique.The results showed that the recombinant hGRN A showed resistance to the growth and division of different kinds of human tumor cells and eventually resulted in the decay of tumor cells.The biological activity of human granulin A?hGRN A?on tumor cells was investigated in this study.It provides an innovative theoretical support for the research of granular protein antitumor drugs.