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

ACS Omega
Fumihiro KawaiRyota Iino

Abstract

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 (X1YSX2X3) were inserted to modify the surface structure of ChiAB. We observed significant amino acid bias at the X3 position in water-soluble, active ChiAB-X1YSX2X3 mutants. Examination of the crystal structure of one active mutant, ChiAB-FYSFV, revealed that the X3 residue plays an important role in structure stabilization.

References

Aug 21, 2003·Journal of Molecular Biology·Marcus D HughesAnna V Hine
Apr 14, 2009·Nature Methods·Daniel G GibsonHamilton O Smith
Feb 4, 2010·Acta Crystallographica. Section D, Biological Crystallography·Wolfgang Kabsch
Feb 4, 2010·Acta Crystallographica. Section D, Biological Crystallography·Paul D AdamsPeter H Zwart
Apr 13, 2010·Acta Crystallographica. Section D, Biological Crystallography·P EmsleyK Cowtan
Jan 14, 2012·Nucleic Acids Research·Yongwei ZhangWinfried Edelmann
Jan 4, 2013·PloS One·Elad Firnberg, Marc Ostermeier
Aug 13, 2013·Journal of the American Chemical Society·Manfred T Reetz
Jun 5, 2014·Nature Communications·Kiyohiko IgarashiMasahiro Samejima
Jun 7, 2016·Scientific Reports·Javier García-NafríaIngo H Greger
Sep 27, 2016·Scientific Reports·Xiao-Fei ZhangYan Feng
Nov 1, 2016·Nature Methods·Emily E WrenbeckTimothy A Whitehead
Dec 24, 2016·Journal of Synchrotron Radiation·Nobuhisa WatanabeMasao Tabuchi
Jan 17, 2017·Biochemistry·Huifang YinSander S van Leeuwen
Sep 29, 2017·Biochemistry and Biophysics Reports·Ken Motohashi
Nov 2, 2017·Physical Chemistry Chemical Physics : PCCP·Akihiko NakamuraRyota Iino

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Methods Mentioned

BETA
PCR
PCRs
electrophoresis

Software Mentioned

XDSGUI
PHENIX
XDS
refine
XDSME

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