Development of a full-length human protein production pipeline

Justin Saul, Brianne Petritis, Sujay Sau, Femina Rauf, Michael Gaskin, Benjamin Ober-Reynolds, Irina Mineyev, Dewey Magee, John Chaput, Ji Qiu, Joshua LaBaer

Research output: Contribution to journalArticle

14 Scopus citations

Abstract

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 Escherichia coli 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 LabChipVR GX microfluidic capillary gel electrophoresis instrument. This automated, HT assay provided both qualitative and quantitative assessment of recombinant protein. E. coli 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 E. coli, 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.

Original languageEnglish (US)
Pages (from-to)1123-1135
Number of pages13
JournalProtein Science
Volume23
Issue number8
DOIs
StatePublished - Aug 2014

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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    Saul, J., Petritis, B., Sau, S., Rauf, F., Gaskin, M., Ober-Reynolds, B., Mineyev, I., Magee, D., Chaput, J., Qiu, J., & LaBaer, J. (2014). Development of a full-length human protein production pipeline. Protein Science, 23(8), 1123-1135. https://doi.org/10.1002/pro.2484