High-resolution sequence-function mapping of full-length proteins

PloS One
Caitlin A KowalskyTimothy A Whitehead

Abstract

Comprehensive sequence-function mapping involves detailing the fitness contribution of every possible single mutation to a gene by comparing the abundance of each library variant before and after selection for the phenotype of interest. Deep sequencing of library DNA allows frequency reconstruction for tens of thousands of variants in a single experiment, yet short read lengths of current sequencers makes it challenging to probe genes encoding full-length proteins. Here we extend the scope of sequence-function maps to entire protein sequences with a modular, universal sequence tiling method. We demonstrate the approach with both growth-based selections and FACS screening, offer parameters and best practices that simplify design of experiments, and present analytical solutions to normalize data across independent selections. Using this protocol, sequence-function maps covering full sequences can be obtained in four to six weeks. Best practices introduced in this manuscript are fully compatible with, and complementary to, other recently published sequence-function mapping protocols.

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Citations

Jul 28, 2016·Technology·Justin R Klesmith, Timothy A Whitehead
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Jul 13, 2019·Protein Engineering, Design & Selection : PEDS·Angélica V Medina-CucurellaTimothy A Whitehead
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Sep 11, 2021·ACS Synthetic Biology·Matthew P DeJongBenjamin J Hackel
Oct 6, 2021·Journal of the American Chemical Society·Michael HeyneJulia M Shifman

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Methods Mentioned

BETA
phage display
fluorescence activated cell sorting
scraping
PCR
Illumina sequencing
fluorescence-activated cell sorting
FACS
protein display

Software Mentioned

QuikChange calculator
Agilent QuikChange
EMPIRIC
Enrich
QuikChange Primer Design
Pfunkel
custom

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