Studies on the expression and processing of human proinsulin derivatives encoded by different DNA constructs

Area of Science:

• Biotechnology
• Molecular Biology
• Protein Engineering

Background:

• Recombinant human insulin production is crucial for diabetes management.
• Optimizing gene expression and protein processing in microbial systems is essential for efficient biopharmaceutical manufacturing.

Purpose of the Study:

• To engineer a synthetic human proinsulin gene for enhanced expression in Escherichia coli.
• To develop a streamlined method for converting expressed proinsulin into mature insulin.
• To evaluate different gene constructs and processing strategies for recombinant insulin production.

Main Methods:

• Synthetic gene design with E. coli preferred codons and N-terminal modifications.
• Construction and expression of multiple proinsulin gene derivatives in E. coli.
• Refolding of expressed inclusion bodies and enzymatic processing for insulin maturation.
• Analysis of gene expression using predictive models.

Main Results:

• Proinsulin derivatives were expressed as inclusion bodies and successfully refolded.
• No significant improvement in expression was observed with gene fusions or multiple gene copies.
• The N-terminal sequence MKR↓F facilitated efficient single-step conversion to insulin using trypsin and carboxypeptidase B.
• Predictive models for protein expression showed no significant correlation with observed results.

Conclusions:

• The engineered MKR↓F linker provides an efficient strategy for the one-step conversion of recombinant proinsulin to insulin.
• Escherichia coli can be utilized for the production of insulin derivatives, with optimization of the processing step being key.
• Further investigation into predictive models for gene expression may be warranted.