SAS: Split antibiotic selection for identifying chaperones that improve protein solubility.

Area of Science:

• Biotechnology
• Molecular Biology
• Protein Engineering

Background:

• Heterologous protein expression in Escherichia coli is crucial for research and industry.
• Achieving soluble and active recombinant proteins in E. coli presents significant challenges.
• Current methods for enhancing protein solubility are often time-consuming and lack a universal solution.

Purpose of the Study:

• To develop a novel genetic system for selecting protein folding factors.
• To link protein solubility directly to cellular viability for efficient screening.
• To create a versatile tool for improving recombinant protein production.

Main Methods:

• Developed a Split Antibiotic Selection (SAS) system using a tripartite fusion with aminoglycoside 7″-phosphotransferase-Ia (APH(7″)).
• Inserted target proteins in-frame within the APH(7″) coding sequence to create a solubility-dependent hygromycin B resistance.
• Utilized a chaperone library for pooled genetic selection to identify solubility-enhancing factors.

Main Results:

• Demonstrated SAS functionality with human mitochondrial Hsp70 ATPase domain and its co-chaperone Hep.
• Showcased that cellular hygromycin B resistance directly correlates with the solubility of the tripartite fusion protein.
• Successfully identified chaperones that improve client protein solubility using pooled SAS screening.

Conclusions:

• The tripartite APH(7″) fusion system effectively links in vivo protein solubility to hygromycin B resistance.
• SAS enables selection of chaperones that enhance the solubility of various client proteins.
• This system streamlines the identification of optimal folding conditions, reducing the need for individual testing.