Heterologous Expression And Purification Of Hagfish Mucus Protein

To realize the application and production of hagfish mucus protein,this experiment successfully expressed hagfish mucus protein,and increased the protein expression and improved its purification method.This experiment tried to supplement the tRNA corresponding to the amino acid that accounts for a high proportion of the amino acid sequence of hagfish mucus protein to increase the protein expression,but it did not achieve the expected effect.Finally,according to codon preference,the hagfish mucus protein gene was optimized to increase the production of target protein.Then,the protein expression conditions of the host bacteria were optimized,and the IPTG concentration,induction time and supplementation amounts of glycine,threonine,and serine were evaluated in single-factor tests.On the basis of single-factor experiments,the response surface experiment was optimized for the addition amount of glycine,threonine,and serine.The results show that the best induction conditions for EsTKa shake flasks are IPTG concentration 0.6 mmol/L,induction for 12 h,and glycine,threonine,and serine added at 90 mg/L,90 mg/L,and 9.91 mg/L,respectively,to achieve the highest protein yield of 153.482 mg/L.The IPTG concentration of EsTKy was 0.8 mmol/L after induction for 12 h,and the amounts of glycine,threonine,and serine were 54 mg/L,9.01 mg/L,11.4 mg/L,respectively.The theoretical best protein yield was 141.97 mg/L.And try to use size exclusion chromatography and nickel column affinity chromatography to purify and isolate the protein.Although a relatively pure protein sample is obtained,the time-consuming and cumbersome operation process is not conducive to the later application and production.Finally,based on the principle of specific self-assembly between proteins,the two proteins were subjected to gradient dialysis,and the gelled assembled protein was selected by the phase separation method to achieve separation and purification.