A versatile modular vector set for optimizing protein expression among bacterial, yeast, insect and mammalian hosts

Area of Science:

• Molecular Biology
• Biotechnology
• Protein Expression

Background:

• Predicting optimal protein expression conditions (host and affinity tag) is challenging.
• Existing expression systems lack universal versatility.
• Need for a streamlined approach to screen diverse expression parameters.

Purpose of the Study:

• To develop a unified and versatile vector set for recombinant protein expression.
• To facilitate rapid screening of protein expression across multiple host systems and affinity tags.
• To enable efficient expression of large and complex proteins.

Main Methods:

• Development of a novel expression cassette with a unified multi-cloning site, affinity tags, and protease cleavage sites.
• Modification of four established expression systems (E. coli, Pichia pastoris, insect, and mammalian cells) into the pONE vector series.
• Utilizing restriction digestion and ligation for seamless gene and cassette element exchange without PCR.

Main Results:

• Successfully applied the pONE vector set for expressing human proteins RASSF1A, Aurora A, and LIMK1.
• Expressed the large 164 kDa protein Rho-associated protein kinase 2 (ROCK2) in Pichia pastoris yeast.
• Demonstrated that yeast-expressed ROCK2 activity is identical to insect cell-produced counterpart, showcasing yeast as a viable alternative for large proteins.

Conclusions:

• The pONE vector set offers significant versatility for recombinant protein expression screening.
• The cost-effective yeast Pichia pastoris system can successfully express large, complex mammalian proteins, rivaling traditional insect cell systems.
• This technology streamlines the discovery of optimal protein expression strategies.