14 Citations (Scopus)

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 - 2014

Fingerprint

Pipelines
Proteins
Escherichia coli
Cell-Free System
Cell Extracts
HeLa Cells
Germ Cells
Triticum
Fusion reactions
Throughput
Microfluidics
Triage
Escherichia coli Proteins
Capillary Electrophoresis
Recombinant Proteins
Proteomics
Electrophoresis
Labeling
Purification
Fluorescence

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Medicine(all)

Cite this

Saul, J., Petritis, B., Sau, S., Rauf, F., Gaskin, M., Ober-Reynolds, B., ... 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

Development of a full-length human protein production pipeline. / Saul, Justin; Petritis, Brianne; Sau, Sujay; Rauf, Femina; Gaskin, Michael; Ober-Reynolds, Benjamin; Mineyev, Irina; Magee, Dewey; Chaput, John; Qiu, Ji; LaBaer, Joshua.

In: Protein Science, Vol. 23, No. 8, 2014, p. 1123-1135.

Research output: Contribution to journalArticle

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, vol. 23, no. 8, pp. 1123-1135. https://doi.org/10.1002/pro.2484
Saul J, Petritis B, Sau S, Rauf F, Gaskin M, Ober-Reynolds B et al. Development of a full-length human protein production pipeline. Protein Science. 2014;23(8):1123-1135. https://doi.org/10.1002/pro.2484
Saul, Justin ; Petritis, Brianne ; Sau, Sujay ; Rauf, Femina ; Gaskin, Michael ; Ober-Reynolds, Benjamin ; Mineyev, Irina ; Magee, Dewey ; Chaput, John ; Qiu, Ji ; LaBaer, Joshua. / Development of a full-length human protein production pipeline. In: Protein Science. 2014 ; Vol. 23, No. 8. pp. 1123-1135.
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