Rational Protein Engineering Guided by Deep Mutational Scanning

Abstract

Sequence–function relationship in a protein is commonly determined by the three-dimensional protein structure followed by various biochemical experiments. However, with the explosive increase in the number of genome sequences, facilitated by recent advances in sequencing technology, the gap between protein sequences available and three-dimensional structures is rapidly widening. A recently developed method termed deep mutational scanning explores the functional phenotype of thousands of mutants via massive sequencing. Coupled with a highly efficient screening system, this approach assesses the phenotypic changes made by the substitution of each amino acid sequence that constitutes a protein. Such an informational resource provides the functional role of each amino acid sequence, thereby providing sufficient rationale for selecting target residues for protein engineering. Here, we discuss the current applications of deep mutational scanning and consider experimental design.

Control of metabolite efflux in microbial cell factories: current advances and future prospects

“…[324,325,392,393]). The opportunities afforded by 'deep mutational scanning' [394][395][396][397][398][399], the coupled deep sequencing, and the 'deep' learning [400,401] of structure(sequence)-activity relationships [324,402] are enormous.…”

Affinity Maturation of a Cyclic Peptide Handle for Therapeutic Antibodies Using Deep Mutational Scanning

“…Another methodological issue that we addressed is how to deal with sampling error. Enrichment ratios are usually calculated by dividing the frequency of a particular mutation in the selected population by the corresponding frequency in the unselected library (17)(18)(19). However, it is easier to enrich rare amino acids than frequent ones.…”

“…Because subtle affinity enhancements may escape detection in classical screens for a select number of "hit" sequences, recently high-throughput protein display technologies have been used in conjunction with deep sequencing before and after selection, a combination loosely referred to as "deep mutational * This work was supported by European Research Council (ERC) Starting scanning" (17)(18)(19). This strategy allows the construction of fitness landscapes of the selected property (i.e.…”

Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis

“…Scanning libraries introduce a single point mutation at different sites in a target gene. Alanine scanning is a traditional approach to identify functionally important residues in enzymes(31) but scanning libraries can also be used to map the local functional neighbourhood of an enzyme (32) or even to generate datasets for deep mutational scanning-guided rational protein design (33). Mutant generation can be laborious when mutations are introduced individually by site-directed mutagenesis.…”