[Yeast (Saccharomyces cerevisiae) secretory expression vector maintained stably in Pro3+ transformants in rich...

Area of Science:

• Biotechnology
• Molecular Biology
• Yeast Genetics

Background:

• Developing efficient methods for foreign gene expression in yeast is crucial for biotechnology.
• Traditional yeast selection often requires specialized media, complicating the process.
• The PRO3 gene's role in yeast auxotrophy offers a potential avenue for simplified selection.

Purpose of the Study:

• To construct a yeast secretory expression vector utilizing the PRO3 gene.
• To evaluate the efficacy of rich medium (YPD) as a selection pressure for yeast transformants.
• To assess the expression level of a foreign gene (beta-HCG) using this novel vector system.

Main Methods:

• Construction of a yeast expression vector (pCBy310) containing the PRO3 gene.
• Insertion of human chorionic gonadotropin beta-subunit (beta-HCG) cDNA into the vector to create pCBy314.
• Transformation of yeast Pro3- auxotroph (MB299-7A) and selection in YPD medium.
• Cultivation under non-optimized conditions to measure beta-HCG expression.

Main Results:

• The recombinant plasmid pCBy314 was successfully constructed and maintained mitotically stable in yeast Pro3- auxotrophs in YPD medium.
• Yeast demonstrated improved growth in YPD, leading to enhanced foreign gene expression.
• Beta-HCG expression reached 650 micrograms/L under the tested conditions.

Conclusions:

• Yeast rich medium (YPD) can serve as an effective selection medium for PRO gene-based vectors.
• The PRO gene system facilitates direct selection in simple, inexpensive rich media, improving yeast transformation efficiency.
• This approach offers a versatile strategy for cloning and expressing foreign genes in yeast, enhancing productivity.