Description

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

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 LabChip[superscript ®] 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.

Downloads
PDF (460.9 KB)

Details

Title
  • Development of a Full-Length Human Protein Production Pipeline
Contributors
Date Created
2014-08-01
Resource Type
  • Text
  • Collections this item is in
    Identifier
    • Digital object identifier: 10.1002/pro.2484
    • Identifier Type
      International standard serial number
      Identifier Value
      0961-8368
    • Identifier Type
      International standard serial number
      Identifier Value
      1469-896X
    Note
    • This is the peer reviewed version of the article, which has been published in final form at http://dx.doi.org/10.1002/pro.2484

    Citation and reuse

    Cite this item

    This is a suggested citation. Consult the appropriate style guide for specific citation guidelines.

    Saul, Justin, Petritis, Brianne, Sau, Sujay, Rauf, Femina, Gaskin, Michael, Ober-Reynolds, Benjamin, Mineyev, Irina, Magee, Mitch, Chaput, John, Qiu, Ji, & LaBaer, Joshua (2014). Development of a full-length human protein production pipeline. PROTEIN SCIENCE, 23(8), 1123-1135. http://dx.doi.org/10.1002/pro.2484

    Machine-readable links