Milk's primary protein component is casein, with κ-casein (κ-CN) comprising roughly 13% of the total casein content in bovine skim milk.κ-CN offers the nutritional benefits typical of milk proteins and holds potential for future applications in total milk synthesis.Moreover, peptides derived from κ-CN hydrolysis exhibit anticoagulant and antithrombotic properties. Corynebacterium glutamicum, a safely produced strain, served as an advantageous platform for edible protein production.This study leveraged C.glutamicum's capability to express heterologous proteins, focusing on the exogenous expression of κ-CN.The κ-CN gene was codon-optimized and synthesized, and the C.glutamicum ATCC13032 strain was utilized to express κ-CN-6×His protein.Optimization efforts targeted the N-terminal solubilization tag, signal peptide, expression host, and fermentation conditions.Results showed that structural analysis revealed fixed disordered regions within κ-CN.Expression of κ-CN was confirmed via SDS-PAGE and western blotting, indicating successful expression in C.glutamicum ATCC13032 strains.Soluble expression was enhanced by incorporating SUMO and FH8 solubilization tags at the N-terminus, with further improvements in protein yield achieved through signal peptide and host screening.This study demonstrates the successful expression of κ-CN in Corynebacterium glutamicum, with optimization strategies enhancing yield and laying the groundwork for future advancements in total milk synthesis.
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