title: High-resolution sequence-function mapping of full-length proteins.
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.
authors: Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing, Michigan, United States of America.
authors: Caitlin A
authors: CA
authors: Kowalsky
authors: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan, United States of America.
authors: Justin R
authors: JR
authors: Klesmith
authors: Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing, Michigan, United States of America.
authors: James A
authors: JA
authors: Stapleton
authors: Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing, Michigan, United States of America.
authors: Vince
authors: V
authors: Kelly
authors: Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing, Michigan, United States of America.
authors: Nolan
authors: N
authors: Reichkitzer
authors: Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing, Michigan, United States of America; Department of Biosystems and Agricultural Engineering, Michigan State University, East Lansing, Michigan, United States of America.
authors: Timothy A
authors: TA
authors: Whitehead
mesh_terms: D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D011506:Proteins / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D011506:Proteins