Deciphering correct strategies for multiprotein complex assembly by co-expression: application to complexes as large...

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• Macromolecular complexes are vital for cellular functions, driving interest in their purification and characterization.
• Co-expression is a key technique for assembling multiprotein complexes, with various systems available for different hosts.
• A lack of comparative knowledge on co-expression strategies hinders efficient complex production.

Purpose of the Study:

• To demonstrate how to delineate correct co-expression strategies for multiprotein complexes.
• To address challenges in stoichiometry imbalance and low yield during complex production.
• To explore the applicability of identified strategies across different expression hosts.

Main Methods:

• Utilized versatile co-expression systems (pET-MCN and pET-MCP series) in Escherichia coli.
• Applied complex-dependent approaches to optimize co-expression.
• Examined parameters influencing co-expression strategies for their host independence.

Main Results:

• Successfully delineated appropriate co-expression strategies for ternary protein complexes.
• Alleviated stoichiometry imbalance and yield problems, preventing production failures.
• Demonstrated host-independent parameters applicable to eukaryotic co-expression systems.
• Enabled the reconstitution of large protein complexes, including the SAGA deubiquitination module and histone octamer, in E. coli.

Conclusions:

• Optimized co-expression strategies are crucial for efficient and successful production of multiprotein complexes.
• The developed approaches enhance protein complex yield and quality, overcoming common production hurdles.
• Findings have implications for co-expression in both prokaryotic and eukaryotic systems, facilitating the study of large molecular machines.