Expression of the human interferon-gamma cDNA in yeast

Area of Science:

• Biotechnology
• Molecular Biology
• Yeast Genetics

Background:

• Human interferon-gamma (IFN-gamma) is a crucial therapeutic protein.
• Efficient and scalable production of IFN-gamma is essential for its clinical application.
• Saccharomyces cerevisiae offers a robust platform for recombinant protein expression.

Purpose of the Study:

• To construct and evaluate yeast expression plasmids for human IFN-gamma production.
• To characterize the expression levels and mRNA features of IFN-gamma in yeast.
• To identify transcription initiation and termination sites for IFN-gamma mRNA.

Main Methods:

• Construction of expression plasmids utilizing the PGK promoter for human IFN-gamma cDNA.
• Transformation of Saccharomyces cerevisiae with engineered plasmids.
• Quantification of biologically active IFN-gamma and analysis of IFN-gamma mRNA levels.
• Localization of transcription initiation and termination sites.

Main Results:

• Achieved high yields of biologically active human IFN-gamma (up to 25 x 10^6 units/liter).
• Observed significantly lower IFN-gamma mRNA levels compared to PGK mRNA.
• Identified the major transcription initiation site 4 base pairs upstream of the PGK mRNA start.
• Localized specific transcription termination and polyadenylation sites within the 3' untranslated region.

Conclusions:

• Successfully established a yeast expression system for high-level production of active human IFN-gamma.
• Characterized key regulatory elements of IFN-gamma mRNA, including initiation and termination sites.
• The findings provide a foundation for optimizing recombinant protein production in yeast.