Work

Kozak sequence libraries for systematically characterizing transgenes across expression levels

Year: 2026

Type: article

Abstract: Typical mammalian overexpression systems test protein sequence variants with little control over expression levels and steady-state protein abundances, hindering interpretations of how protein sequence and expression converge to yield phenotypic outcomes. We explored the translation initiation sequence, commonly referred to as the Kozak sequence, as a means to systematically modulate protein steady-state abundance and cellular function. We performed sort-seq on a randomized library of the 6 nt preceding the start codon, amounting to 4042 sequences, with a single sequence integrated at a shared genomic integration site in each HEK 293T cell. Calibrating the scores revealed a ∼100-fold range of protein steady-state abundances possible through manipulation of the Kozak sequence. We identified human germline variants with predicted expression-reducing Kozak substitutions in disease-associated genes. Modulating the cell surface abundance of the host cell receptor ACE2 controlled the rate at which those cells became infected by SARS-like coronavirus spike pseudotyped particles. We demonstrated the potential of the approach by simultaneously testing Kozak libraries with a small panel of coding variants for ACE2 and STIM1. This approach lays the methodological groundwork for linking the causal relationships between protein sequence, abundance, and functional outcome.

Source: Nucleic Acids Research

DOI: 10.1093/nar/gkag728

Authors: Nidhi Shukla, Nisha D Kamath, John C Snell, Anna M Bruchez, Kenneth A Matreyek

Institution: Case Western Reserve University

Language: English

Topic: RNA and protein synthesis mechanisms

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

SDG: Good health and well-being

Open Access status: gold

Estimated APC paid: $3,992