Mutation of the proteolytic cleavage site enhances stability and yield of recombinant hepatitis C virus core protein in bacterial cells.

The Hepatitis C virus (HCV) core protein is crucial for viral assembly and pathogenesis; however, structural studies have been hampered by its instability and historical reliance on denaturing purification methods. Although a recent non-denaturing purification method for an E. coli-expressed HCV core 1-164 protein enabled initial characterization, the purified protein exhibited poor long-term stability owing to spontaneous proteolytic cleavage. This study aimed to identify the cleavage sites responsible for this degradation and to engineer a more stable variant. N-terminal sequencing of the degraded fragments revealed two major cleavage sites between the residues Tyr35-Leu36 and Arg47-Thr48. On the basis of these findings, we designed mutants with alanine substitutions at these sites. The combined Pro1/2 (L36A/L37A/R47A/T48A/T49A/R50A), was expressed and purified. Compared to the wild-type protein, the Pro1/2 mutant demonstrated a markedly higher yield during purification and significantly improved stability during storage at 4 °C, retaining approximately 60% of the intact protein after five days versus 20% for the wild-type. Importantly, the mutations did not alter the elution profile of the protein by size-exclusion chromatography, suggesting that the overall structure was preserved, and in silico epitope prediction indicated a minimal impact on antigenicity. The enhanced stability and yield of this Pro1/2 mutant provides a robust platform for future high-resolution structural analyses of the HCV core protein and supports its potential use in the development of therapeutics and vaccines.