High expression of synthetic human interferon-gamma cDNA in E. coli

Area of Science:

• Molecular Biology
• Biotechnology
• Protein Expression

Background:

• Efficient production of therapeutic proteins like human interferon-gamma (IFN-gamma) is crucial.
• Optimizing gene expression in bacterial hosts such as E. coli requires careful consideration of various genetic elements.

Purpose of the Study:

• To synthesize human IFN-gamma cDNA with optimized codon usage for E. coli.
• To investigate the impact of varying spacing between the Shine-Dalgarno (SD) sequence and the ATG start codon on protein expression levels.
• To identify expression plasmids that yield high levels of recombinant IFN-gamma.

Main Methods:

• Synthesis of human IFN-gamma cDNA with E. coli-favored codons.
• Construction of nine expression plasmids with distinct SD-ATG spacing.
• Analysis of free energy (ΔG°f298) associated with secondary structures in the translation initiation region (TIR).
• Quantification of IFN-gamma yield in E. coli.

Main Results:

• Expression plasmids exhibited varying free energies in their TIR secondary structures.
• Plasmid pLY4-gamma 5 demonstrated exceptionally high IFN-gamma yield, constituting 60-80% of total bacterial proteins.
• This level of expression is significantly higher than previously reported in literature.

Conclusions:

• Optimal spacing between the SD sequence and ATG, favorable TIR secondary structure stability (ΔG°f298), and codon usage are critical for high-level IFN-gamma expression in E. coli.
• The developed expression system offers a highly efficient method for producing recombinant human IFN-gamma.