Increasing recombinant protein production in E. coli via FACS-based selection of N-terminal coding DNA libraries

Area of Science:

• Biotechnology
• Molecular Biology
• Protein Engineering

Background:

• N-terminal sequences influence recombinant protein expression in Escherichia coli.
• Previous methods for N-terminal sequence optimization were limited and construct-specific.
• A universally applicable method for enhancing protein yield is needed.

Purpose of the Study:

• To develop and implement a novel strategy for increasing recombinant protein production yield.
• To optimize N-terminal sequences using a directed evolution approach.
• To identify sequence modifications that broadly enhance protein expression.

Main Methods:

• Employed a directed evolution methodology utilizing DNA libraries for N-terminal sequences.
• Screened a large number of diversified sequences to identify optimal N-termini.
• Fused a Green Fluorescent Protein (GFP) reporter gene to the target construct.
• Utilized Fluorescence-Activated Cell Sorting (FACS) to select for high-producing cells.

Main Results:

• Successfully increased the production yield of soluble recombinant proteins by over 30-fold for multiple constructs.
• Demonstrated the effectiveness of directed evolution for N-terminal sequence optimization.
• Identified specific N-terminal sequences that enhance protein expression in Escherichia coli.

Conclusions:

• The developed directed evolution approach is a powerful tool for enhancing recombinant protein yield.
• N-terminal sequence optimization is a critical factor for improving protein production.
• This method offers a broadly applicable strategy for increasing protein expression in Escherichia coli.