2026-05-22T14:02:44Zhttps://keep.lib.asu.edu/oai/requestoai:keep.lib.asu.edu:node-1295372025-05-20T15:17:28Zoai_pmh:alloai_pmh:repo_items129537
https://hdl.handle.net/2286/R.I.27051
<p>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</p>
10.1002/pro.2484
0961-8368
1469-896X
http://rightsstatements.org/vocab/InC/1.0/
2014-08-01
2015-08-01T17:26:48
13 pages
eng
Saul, Justin
Petritis, Brianne
Sau, Sujay
Rauf, Femina
Gaskin, Michael
Ober-Reynolds, Benjamin
Mineyev, Irina
Magee, Mitch
Chaput, John
Qiu, Ji
LaBaer, Joshua
Biodesign Institute
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
<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 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.</p>
Text
Development of a Full-Length Human Protein Production Pipeline