Plasmid-Based One-Pot Saturation Mutagenesis and Robot-Based Automated Screening for Protein Engineering

Area of Science:

• Protein engineering
• Molecular biology
• Biochemistry

Background:

• Plasmid-based one-pot saturation mutagenesis and robot-based automated screening are powerful tools for protein engineering.
• Assessing nucleotide and amino acid biases is crucial for optimizing mutagenesis strategies.
• Green fluorescent protein (GFP) and chitinase fusion proteins (ChiAB) are common models for studying protein function and engineering.

Purpose of the Study:

• To evaluate a combined method of saturation mutagenesis and automated screening for protein engineering.
• To identify biases in nucleotide and amino acid incorporation during mutagenesis.
• To engineer a water-soluble and active fusion protein of chitinases ChiA and ChiB (ChiAB).

Main Methods:

• Plasmid-based one-pot saturation mutagenesis was employed.
• Robot-based automated screening was utilized for high-throughput analysis.
• Deep sequencing was performed to analyze nucleotide ratios in primers.
• NNB and NNN codon strategies were compared.
• Specific amino acid insertions (tyrosine and serine) were introduced into the ChiAB surface (X1YSX2X3).

Main Results:

• Gain-of-function mutant ratios were not significantly different from expected values, despite supplier-dependent nucleotide biases in primers.
• Deep sequencing revealed significant nucleotide biases, with NNB showing less bias than NNN.
• Active, water-soluble ChiAB-X1YSX2X3 mutants exhibited significant amino acid bias at the X3 position.
• Crystal structure analysis of ChiAB-FYSFV indicated the X3 residue's critical role in stabilizing the protein structure.

Conclusions:

• The evaluated protein engineering method is effective for generating functional proteins.
• Understanding and mitigating primer nucleotide biases is important for mutagenesis efficiency.
• Specific surface residues, like those at the X3 position, are critical for the stability and function of engineered proteins.