Preparation and activity,stability detection and analysis of Neuritin wild-type protein
Objective To prepare unlabeled Neuritin wild-type(WT)protein that meets the structural requirements of the pharmacopoe-ia,and complete its identification and analysis of its form,activity,and stability.Methods Neuritin WT yeast recombinants conforming to pharmacopoeia structure were constructed by gene recombination technology,and the high-expression strains was screened and iden-tified by SDS-PAGE reduction electrophoresis and Western blot.The unlabeled Neuritin WT protein purification method was estab-lished.The purified proteins were identified by SDS-PAGE reduction electrophoresis for molecular weight,Western blot for immunoge-nicity,SEC-HPLC for fine identification,and host DNA and host protein residues for quality inspection.The existence form was identi-fied by SDS-PAGE non-reduction electrophoresis.The activity of Neuritin WT purified protein was detected and analyzed by establis-hing a method for identifying the activity of recombinant Neuritin protein in accordance with the requirements of the pharmacopoeia,and the stability of Neuritin WT purified protein at different times at 37℃was observed.Results Neuritin WT purified protein with a purity of more than 98%was prepared,the relative molecular weight of protein was 9.7 kDa,the residual amount of host DNA was 0.009 4 ng/agent and the residual amount of host protein accounted for 0.000 8%.The purified protein was detected in the form of monomer and dimer.The activity identification results indicate that the protein has biological activity to maintain neuronal survival;The degrada-tion rate within 3 days at 37℃is less than 10%,and the half-life is not reached within 7 days(the protein degradation rate is less than 35%).Conclusion Neuritin WT unlabeled protein with purity up to 98%,impurity content and structure meeting pharmacopoeia requirements was successfully prepared.The existence form of Neuritin WT protein was analyzed,and it was confirmed that the protein has good biological activity and stability.Neuritin provides material basis for further functional,pharmaceutical and biological products development.
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