Proteome profiling and vector yield optimization in a recombinant adeno-associated virus-producing yeast model

Area of Science:

• Biotechnology
• Molecular Biology
• Proteomics

Background:

• Recombinant adeno-associated viral (rAAV) vector production in yeast shows promise but suffers from low yields.
• Low yields correlate with inefficient AAV DNA encapsidation.
• Understanding yeast host cell responses is crucial for optimizing rAAV production.

Purpose of the Study:

• To investigate proteomic changes in yeast during rAAV production.
• To identify host proteins influencing rAAV vector yields.
• To enhance rAAV production through targeted protein overexpression.

Main Methods:

• Mass spectrometry-based proteomic profiling of rAAV-producing yeast versus controls.
• Gene ontology and network interaction analyses of differentially expressed proteins.
• Functional validation via overexpression of selected host proteins in yeast and insect cells.

Main Results:

• Proteomic analysis revealed significant changes in yeast proteins related to protein folding, amino acid metabolism, gluconeogenesis, and stress response.
• Overexpression of specific proteins (SSA1, SSE1, SSE2, CCP1, GTT1, RVB2) in yeast increased rAAV vector yields up to threefold.
• Overexpression of chaperone proteins (HSP40, HSC70) in insect cells enhanced volumetric vector yield by 50%.

Conclusions:

• Proteomic analysis provides insights into yeast cellular responses during rAAV production.
• Targeted overexpression of identified host proteins, particularly chaperones, can significantly improve rAAV vector yields.
• Proteomic-driven optimization is a valuable strategy for enhancing recombinant viral vector production systems.