期刊
PROTEIN SCIENCE
卷 23, 期 8, 页码 1123-1135出版社
WILEY
DOI: 10.1002/pro.2484
关键词
human protein; cell-free protein expression; in vitro transcription translation; Escherichia coli; E. coli expression; wheat germ extract; HeLa cell extract; microfluidic capillary gel electrophoresis; high throughput; protein expression; protein purification; high throughput expression analysis; full-length protein; HaloTag; SIFT
资金
- NIH [U54DK093449]
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 (R) 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 mu g 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 mu g and >= 90% purity. Based on these observations, we have developed a triage strategy for producing full-length human proteins in these three expression systems.
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