title: Development of a full-length human protein production pipeline.
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 LabChip(®) 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.
authors.affiliation: Virginia G. Piper Center for Personalized Diagnostics, Biodesign Institute, Arizona State University, Tempe, Arizona, 85287-6401.
authors.forename: Justin
authors.initials: J
authors.lastname: Saul
authors.forename: Brianne
authors.initials: B
authors.lastname: Petritis
authors.forename: Sujay
authors.initials: S
authors.lastname: Sau
authors.forename: Femina
authors.initials: F
authors.lastname: Rauf
authors.forename: Michael
authors.initials: M
authors.lastname: Gaskin
authors.forename: Benjamin
authors.initials: B
authors.lastname: Ober-Reynolds
authors.forename: Irina
authors.initials: I
authors.lastname: Mineyev
authors.forename: Mitch
authors.initials: M
authors.lastname: Magee
authors.forename: John
authors.initials: J
authors.lastname: Chaput
authors.forename: Ji
authors.initials: J
authors.lastname: Qiu
authors.forename: Joshua
authors.initials: J
authors.lastname: LaBaer
mesh_terms: D005453:Fluorescence; D006367:HeLa Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D014176:Protein Biosynthesis; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D040901:Proteomics
chemical_list: D011506:Proteins
keywords: E. coli expression; Escherichia coli; HaloTag; HeLa cell extract; SIFT; cell-free protein expression; full-length protein; high throughput; high throughput expression analysis; human protein; in vitro transcription translation; microfluidic capillary gel electrophoresis; protein expression; protein purification; wheat germ extract