Development of a full‐length human protein production pipeline. (2nd June 2014)

• Record Type: Journal Article
• Authors: Saul, Justin; Petritis, Brianne; Sau, Sujay; Rauf, Femina; Gaskin, Michael; Ober‐Reynolds, Benjamin; Mineyev, Irina; Magee, Mitch; Chaput, John; Qiu, Ji; LaBaer, Joshua
• Is Part Of: Protein science. Volume 23:Number 8(2014:Aug.)
• Page Start: 1123
• Page End: 1135
• Publication Date: 2014-06-02
• Subjects: Proteins -- Periodicals; 572.6
• DOI: 10.1002/pro.2484
• Languages: English
• ISSNs: 0961-8368

Abstract

<abstract abstract-type="main"> <title>Abstract</title> <p>There are many proteomic applications that require large collections of purified protein, but parallel production of large numbers of different proteins remains a very challenging task. To help meet the needs of the scientific community, we have developed a human protein production pipeline. Using high‐throughput (HT) methods, we transferred the genes of 31 full‐length proteins into three expression vectors, and expressed the collection as N‐terminal HaloTag fusion proteins in <italic>Escherichia coli</italic> and two commercial cell‐free (CF) systems, wheat germ extract (WGE) and HeLa cell extract (HCE). Expression was assessed by labeling the fusion proteins specifically and covalently with a fluorescent HaloTag ligand and detecting its fluorescence on a LabChip<sup>®</sup> GX microfluidic capillary gel electrophoresis instrument. This automated, HT assay provided both qualitative and quantitative assessment of recombinant protein. <italic>E. coli</italic> was only capable of expressing 20% of the test collection in the supernatant fraction with ≥20 μg yields, whereas CF systems had ≥83% success rates. We purified expressed proteins using an automated HaloTag purification method. We purified 20, 33, and 42% of the test collection from <italic>E. coli</italic>, WGE, and HCE, respectively, with yields ≥1 μg and ≥90% purity. Based on these observations, we have developed a triage strategy for producing full‐length human proteins in these three expression systems.</p> </abstract>