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We evaluated a method for protein engineering using plasmid-based one-pot saturation mutagenesis and robot-based automated screening. When the biases in nucleotides and amino acids were assessed for a loss-of-function point mutation in green fluorescent protein, the ratios of gain-of-function mutants were not significantly different from the expected values for the primers among the three different suppliers. However, deep sequencing analysis revealed that the ratios of nucleotides in the primers were highly biased among the suppliers. Biases for NNB were less severe than for NNN. We applied this method to screen a fusion protein of two chitinases, ChiA and ChiB (ChiAB). Three NNB codons as well as tyrosine and serine (X 1 YSX 2 X 3 ) were inserted to modify the surface structure of ChiAB. We observed significant amino acid bias at the X 3 position in water-soluble, active ChiAB-X 1 YSX 2 X 3 mutants. Examination of the crystal structure of one active mutant, ChiAB-FYSFV, revealed that the X 3 residue plays an important role in structure stabilization.

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