Expression,Purification And Activity Analysis Of Recombinant Human ProNGF In CHO Cells

proNGF is a precursor of nerve growth factor(NGF),which has biological activity to induce nerve cell apoptosis,and is closely associated with Alzheimer’s disease,Parkinson’s disease,and a variety of diseases caused by nerve cell damage.Natural proNGF is rapidly cleaved by furin protease to form NGF,which has a short half-life and rarely exists in a stable form.In order to provide stable proNGF for the pathogenesis of a variety of neurodegenerative diseases,this study carried out point mutations on the amino acid sequence of human proNGF using an online prediction tool,designed and constructed recombinant human proNGF expression vector p CHO-SU1-proNGF,and transferred it into CHO cells for expression.After Puromycin and MTX pressure screening,monoclonal expression cell lines were obtained by limited dilution,and their productivity was evaluated,further screening candidate CHO engineering cell lines expressing recombinant human proNGF with stable and high efficiency was conducted.The feeding fermentation process of candidate CHO engineering cell lines was optimized by large-scale fermentation technology,and the feeding fermentation process was determined.The target protein was isolated and purified by centrifugation,affinity chromatography and ion exchange chromatography,and then identified.Finally,the biological activity of recombinant human proNGF was determined by SH-SY5 Y cell activity assay.The results showed that replacing arginine at position 52 with leucine and arginine at position 121 with alanine could destroy the cleavage site of furin protease and avoid the cleavage of precursor protein,so as to obtain stable proNGF.Through the optimization of feeding process,proNGF production can be increased by 151.956%;The purity of target protein was 93.61% after purification of supernatant by large-scale fermentation.The results showed that the concentration of recombinant human proNGF was positively correlated with the inhibitory rate of cell growth,which indicated that the recombinant human proNGF had the biological activity of inhibiting the growth of nerve cells.In this study,stable recombinant human proNGF with biological activity was prepared,which provided a certain experimental basis for proNGF related disease mechanism research and further application research.