To realize the application and production of hagfish mucus protein, this experiment increased the protein expression and improved its purification method. 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, with the supplementation of glycine,
threonine, and serine as independent variables, the target protein yield was the response value. According to the Box-Behnken central combination design principle of the response surface method, the influence of the respective variables and their interaction on the hagfish mucus protein yield
were studied, and the induction conditions were optimized through a combination of Design-Expert software and response surface analysis. The results show that the best induction conditions for EsTKα 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 EsTKγ 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, and 11.4 mg/L,
respectively. The theoretical best protein yield was 141.97 mg/L. 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.