Codon Optimization And Expression In Pichia Pastoris Of The Sea Cucumber Lysozyme

Lysozyme(lysozyme,EC3.2.1.17),is also known as muramidase(muramidase).Based on the immunological characteristics and the spatial structure of the catalytic activity,lysozyme was divided into six categories.At present,most research were focused on the c-type human lysozyme and c-type and i-type lysozyme aquatic animals.It had demonstrated that i-type lysozyme had antibacterial effects against Gram-positive bacteria and Gram-negative bacteria.So i-type lysozyme has a very wide range of applications instead of antibiotics.But so far it had found some problems in the process of the expression for the recombinant enzyme cucumber.It was found that protein was prone to inclusion,and easy to inactivation in the E.coli system.Our research had showed that the sea cucumber lysozyme could express in the soluble form,but the expression level was very low.To solve this problem,we will optimize the sea cucumber lysozyme gene codon to improve the expression level.We adjusted the genetic code to optimizate fermentation and improve the yield based on the the Codon preference and AT content of the active binding experiment.In this study,the recombinant cloning vector pMD18-T-SjLys' was digested by EcoR I and Not I,and built the expression vector pPIC9K-SjLys'.We transformed pPIC9K-SjLys' into Pichia pastoris GS115.MD and MM agar meda were used to screen slow phenotype with methanol,and then through the YPD medium containing G418 1mg/mL-4mg/mL to screenin strains with highly expression.256 strains were screened out,the product of the fermentation broth by TCA sediment was detected by SDS-PAGE.Experiments show that,by the sea cucumber lysozyme gene codon optimization,the expression of a significant improvement could be achieved.Pichia pastoris expression out of the sea cucumber lysozyme can inhibit Gram-positive and Gram-negative indicator bacteria activity.In this study,we successfully got sea cucumber lysozyme expressed by Pichia pastoris.The expression level of sea cucumber lysozyme was enhanced by the gene codon optimization.Our research studies the structure and function of lysozyme and purification which laid the foundation for future large-scale industrial production.