Effect of Neuritin C56S mutant protein on its structure,activity and thermal stability
Objective The disulfide bonds,formed by cysteine residues in protein molecules,serve as a crucial stabilizing force for maintaining protein structure.The study is to observe the effects of cysteine at site 56 on the structure,activity and thermal stability of Neuritin protein by the mutating Cys to Ser at 56 site.Methods Through the analysis of experimental results and the predicted structure from bioinformatics,the mutation sites were determined.The pPIC9K-Neuritin C56S mutant shuttle plasmid was constructed by over-lapping extension PCR.After electrotransfer to Pichia pastoris GS115,the mutant yeast recombinant was successfully obtained with G418 resistance screening,genotype identification and high expression screening.Finally,the strain with the highest expression level was gained.The Neuritin C56S mutant protein was purified using our established purification process,and the purified protein was i-dentified by SDS-PAGE electrophoresis coomassie brilliant blue staining and Western blot.Subsequently,the existence form of the mu-tant protein was identified by SDS-PAGE non-reducing electrophoresis coomassie brilliant blue staining.Using the biological activity verification method of recombinant human Neuritin protein established by our research group based on the pharmacopoeia,the activity of the Neuritin C56S mutant protein was detected by observing the survival of hippocampal neurons HT22 cells.The degradation of the mutant protein at 37 ℃ and different time points was determined.Results The Pichia pastoris expression system of Neuritin with C56S mutant was constructed,and the recombinant strain of Pichia Pastoris with the highest expression was selected,and the C56S mutant protein of Neuritin with purity of 98％was obtained.Compared with the unmodified Neuritin protein,the mutant protein showed none multimer and more dimers,indicating that there was only one free cysteine residue in the Neuritin C56S mutant protein monomer,which can only form dimers,but not multimers.Therefore,it can be inferred that the other intra-chain disulfide bonds are stable,and the unstable intra-chain disulfide bond is C56-C4.Meantime,since there is only one free cysteine,its dimer can only form an inter-chain disulfide bond,so the degradation of Neuritin C56S mutant protein was significantly increased at 37℃for 14 days,further indi-cating that C56-C4 in Neuritin protein is an unstable intrachain disulfide bond.Conclusion The intra-chain disulfide bonds between C56 and C4 in Neuritin protein is unstable.Point mutations in Neuritin C56S affect its natural structural form and further affect its ac-tivity and thermal stability.
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