Codon preference optimization increases prokaryotic cystatin C expression

Area of Science:

• Biotechnology
• Molecular Biology
• Genetic Engineering

Background:

• Gene expression efficiency in prokaryotic systems is crucial for protein production.
• Human cystatin C (cysC) expression in Escherichia coli (E. coli) can be limited by codon usage.
• Optimizing codon preference is a strategy to enhance recombinant protein yield.

Purpose of the Study:

• To improve the expression of the human cystatin C (cysC) gene in Escherichia coli (E. coli) through codon optimization.
• To evaluate the impact of codon optimization on cysC protein yield and purity.
• To assess the feasibility of using codon optimization for commercial production.

Main Methods:

• The human cysC gene sequence was redesigned using E. coli codon preferences, maintaining the original amino acid sequence.
• Codon-optimized cysC (co-cysC) and wild-type cysC (wt-cysC) were cloned into a pET-30a plasmid and transformed into E. coli BL21.
• Recombinant protein expression levels and purity were analyzed before and after optimization.

Main Results:

• Codon optimization increased recombinant protein expression from 10% to 46% of total protein.
• Purified recombinant CysC achieved a purity level exceeding 95%.
• Biological activity of CysC was confirmed post-optimization.

Conclusions:

• Codon optimization is an effective strategy for significantly enhancing cysC gene expression in E. coli.
• The improved expression levels and high purity are suitable for potential commercial production.
• This approach can be applied to other genes for improved recombinant protein manufacturing.