Systematic optimization of active protein expression using GFP as a folding reporter

Area of Science:

• Biotechnology
• Protein Expression
• Biochemistry

Background:

• Recombinant human proteins often exhibit poor solubility and incorrect folding when expressed in heterologous hosts like E. coli.
• Interleukin-18 (IL-18) is a crucial cytokine implicated in autoimmune diseases, but its production faces challenges due to inclusion body formation.
• Previous attempts to optimize IL-18 expression yielded low amounts of active protein.

Purpose of the Study:

• To develop an efficient screening system for optimizing the expression conditions of human Interleukin-18 (hIL-18) in Escherichia coli.
• To overcome the common issue of insoluble protein aggregates (inclusion bodies) during recombinant protein production.
• To enhance the yield of correctly folded, active hIL-18 for potential therapeutic applications.

Main Methods:

• A folding reporter system utilizing green fluorescent protein (GFP) was employed to screen various expression conditions.
• Systematic screening facilitated the identification of optimal parameters for hIL-18 expression.
• The system allowed for high-throughput evaluation of different expression variables.

Main Results:

• The GFP reporter system efficiently screened numerous expression conditions for hIL-18.
• Optimized conditions led to a significant increase in the yield of active, correctly folded hIL-18.
• The method successfully mitigated the problem of inclusion body formation.

Conclusions:

• A GFP-based folding reporter system provides an effective strategy for optimizing recombinant protein expression.
• This approach can be broadly applied to enhance the production of other challenging proteins in various host organisms.
• The developed method holds promise for improving the manufacturing of therapeutic proteins like IL-18.