Heterologous Expression and Purification of Hagfish Mucus Protein

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.

Optimization of culture conditions for the expression of three different insoluble proteins in Escherichia coli

Recombinant protein expression for structural and therapeutic applications requires the use of systems with high expression yields. Escherichia coli is considered the workhorse for this purpose, given its fast growth rate and feasible manipulation. However, bacterial inclusion body formation remains a challenge for further protein purification. We analyzed and optimized the expression conditions for three different proteins: an anti-MICA scFv, MICA, and p19 subunit of IL-23. We used a response surface methodology based on a three-level Box-Behnken design, which included three factors: post-induction temperature, post-induction time and IPTG concentration. Comparing this information with soluble protein data in a principal component analysis revealed that insoluble and soluble proteins have different optimal conditions for post-induction temperature, post-induction time, IPTG concentration and in amino acid sequence features. Finally, we optimized the refolding conditions of the least expressed protein, anti-MICA scFv, using a fast dilution protocol with different additives, obtaining soluble and active scFv for binding assays. These results allowed us to obtain higher yields of proteins expressed in inclusion bodies. Further studies using the system proposed in this study may lead to the identification of optimal environmental factors for a given protein sequence, favoring the acceleration of bioprocess development and structural studies.