[Expression of Pichia pastoris tRNAProCCG and its function]

Area of Science:

• Molecular Biology
• Biotechnology
• Protein Synthesis

Background:

• Transfer ribonucleic acid (tRNA) is crucial for protein synthesis.
• Rare codons can limit the expression of exogenous genes.
• Understanding the impact of rare tRNAs on gene expression is essential for optimizing recombinant protein production.

Purpose of the Study:

• To investigate the effect of rare tRNA expression on exogenous gene expression in Pichia pastoris.
• To construct a co-expression system for rare tRNA genes and exogenous genes.
• To enhance the expression of genes repressed by rare codons.

Main Methods:

• Constructed a co-expression system in Pichia pastoris.
• Introduced a repressor region with continuous proline rare codon CCG into the GFP gene to reduce expression.
• Cointegrated tRNAProCCG gene with the repressed GFP gene using plasmids pPIC9K and pFLDα.
• Co-expressed NFATc3T-GFP fusion gene and tRNAProCCG gene.

Main Results:

• Co-integration of tRNAProCCG gene increased repressed GFP expression by 4.9% and 12.5% through different plasmid strategies.
• Co-expression of NFATc3T-GFP fusion gene and tRNAProCCG gene resulted in a 21.3% increase in fusion protein expression.
• Confirmed tRNAProCCG as a rare tRNA in Pichia pastoris GS115.
• Demonstrated significant increase in expression of repressed heterologous genes containing continuous rare codon CCG.

Conclusions:

• Co-expression of tRNAProCCG gene with heterologous genes containing rare CCG codons significantly enhances the expression of repressed genes.
• The developed co-expression system is effective for improving protein yields.
• This system can be utilized for screening and identifying other rare tRNAs in Pichia pastoris.