Optimizing Granulocyte Colony-Stimulating Factor Transcript for Enhanced Expression in Escherichia coli.

Area of Science:

• Biotechnology
• Molecular Biology
• Protein Expression

Background:

• Granulocyte colony-stimulating factor (G-CSF) is a crucial hematopoietic growth factor for treating neutropenia and aiding bone marrow transplantation.
• Recombinant G-CSF is produced in Escherichia coli, but achieving high expression yields for therapeutic applications remains a challenge.
• Previous strategies for enhancing protein expression include codon optimization and promoter selection, but these have shown limited success for G-CSF.

Purpose of the Study:

• To investigate the reasons for low G-CSF protein expression in E. coli despite codon optimization.
• To identify and overcome translational barriers in G-CSF mRNA.
• To develop a strategy for significantly increasing recombinant G-CSF production.

Main Methods:

• Analysis of the 5' end mRNA secondary structure of native and codon-optimized G-CSF transcripts.
• Introduction of translationally silent mutations in the first 24 nucleotides of the G-CSF transcript to disrupt stable secondary structures.
• Comparison of G-CSF protein expression levels in E. coli between native and modified constructs.

Main Results:

• Stable secondary structures were identified at the 5' end of both native and codon-optimized G-CSF mRNA.
• Disrupting these secondary structures via silent mutations led to a remarkable 60-fold increase in G-CSF protein expression.
• The protein sequence of G-CSF remained unaffected by the introduced mutations.

Conclusions:

• The 5' mRNA secondary structure significantly impedes G-CSF translation in E. coli.
• Targeted disruption of these structures offers a potent strategy for enhancing G-CSF expression.
• This approach provides a roadmap for optimizing other therapeutic protein transcripts for improved production in E. coli.