High-level expression of a gene encoding the human complement factor C5a in Escherichia coli.

Area of Science:

• Molecular Biology
• Protein Expression
• Biotechnology

Background:

• Poor expression of synthetic C5a gene in Escherichia coli was observed.
• Investigating mRNA structure and C5a product stability in E. coli is crucial for improving expression.

Purpose of the Study:

• To identify reasons for poor C5a gene expression in E. coli.
• To enhance C5a expression and stability in E. coli.

Main Methods:

• Varied mRNA 5' end sequences and ribosome-binding sites.
• Assessed C5a stability in wild-type and protease-deficient E. coli strains, including an htpR mutant.
• Optimized expression vector, regulatory regions, host strain, growth conditions, and recovery methods.

Main Results:

• C5a exhibited rapid degradation (3-5 min half-life) in wild-type E. coli.
• C5a stability increased significantly (20 min half-life) in an htpR mutant strain with proteolytic deficiency.
• Achieved a 300-fold increase in C5a levels, reaching approximately 3% of total cellular protein.

Conclusions:

• mRNA structure and host strain proteases significantly impact C5a expression and stability.
• Utilizing an htpR mutant and optimizing expression parameters can dramatically enhance C5a production in E. coli.
• This study provides a robust strategy for high-level recombinant protein production in E. coli.