A cell-free protein synthesis system for high-throughput proteomics

TLDR

The wheat‑seed‑based cell‑free system offers advantages over other commonly used cell‑free expression platforms. The study presents a cell‑free system for high‑throughput synthesis and screening of gene products. The system maximizes yield and throughput by optimizing mRNA UTRs, designing a large‑scale expression vector, developing a PCR‑based DNA construction strategy, and validating protein folding with enzymatic assays and NMR. The system achieves high‑yield expression, maintains productive translation for 14 days, can translate at least 50 genes in parallel producing 0.1–2.3 mg protein per person in 2 days, bypasses time‑consuming cloning steps, and is amenable to robotic automation.

Abstract

We report a cell-free system for the high-throughput synthesis and screening of gene products. The system, based on the eukaryotic translation apparatus of wheat seeds, has significant advantages over other commonly used cell-free expression systems. To maximize the yield and throughput of the system, we optimized the mRNA UTRs, designed an expression vector for large-scale protein production, and developed a new strategy to construct PCR-generated DNAs for high-throughput production of many proteins in parallel. The resulting system achieves high-yield expression and can maintain productive translation for 14 days. Additionally, in the integration of a PCR-directed system for template creation, at least 50 genes can be translated in parallel, yielding between 0.1 and 2.3 mg of protein by one person within 2 days. Assessment of correct protein folding by the products of this high-throughput protein-expression system were performed by enzymatic assays of kinases and by NMR spectroscopic analysis. The cell-free system, reported here, bypasses many of the time-consuming cloning steps of conventional expression systems and lends itself to a robotic automation for the high-throughput expression of proteins.