title: High-throughput instant quantification of protein expression and purity based on photoactive yellow protein turn off/on label.
abstract: Quantifying the concentration and purity of a target protein is essential for high-throughput protein expression test and rapid screening of highly soluble proteins. However, conventional methods such as PAGE and dot blot assay generally involve multiple time-consuming tasks requiring hours or do not allow instant quantification. Here, we demonstrate a new method based on the Photoactive yellow protein turn Off/On Label (POOL) system that can instantly quantify the concentration and purity of a target protein. The main idea of POOL is to use Photoactive Yellow Protein (PYP), or its miniaturized version, as a fusion partner of the target protein. The characteristic blue light absorption and the consequent yellow color of PYP is absent when initially expressed without its chromophore, but can be turned on by binding its chromophore, p-coumaric acid. The appearance of yellow color upon adding a precursor of chromophore to the co-expressed PYP can be used to check the expression amount of the target protein via visual inspection within a few seconds as well as to quantify its concentration and purity with the aid of a spectrometer within a few minutes. The concentrations measured by the POOL method, which usually takes a few minutes, show excellent agreement with those by the BCA Kit, which usually takes ∼1 h. We demonstrate the applicability of POOL in E. coli, insect, and mammalian cells, and for high-throughput protein expression screening.
authors.affiliation: Department of Chemistry, KAIST, Daejeon, 305-701, Republic of Korea.
authors.forename: Youngmin
authors.initials: Y
authors.lastname: Kim
authors.forename: Prabhakar
authors.initials: P
authors.lastname: Ganesan
authors.forename: Hyotcherl
authors.initials: H
authors.lastname: Ihee
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D003124:Colorimetry; D003373:Coumaric Acids / Q000378:metabolism; D004926:Escherichia coli; D057166:High-Throughput Screening Assays; D006863:Hydrogen-Ion Concentration; D020283:Photoreceptors, Microbial / Q000378:metabolism*; D011422:Propionates; D011485:Protein Binding; D014176:Protein Biosynthesis; D011506:Proteins / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D061987:Sf9 Cells; D013056:Spectrophotometry, Ultraviolet; D018411:Spodoptera
chemical_list: D001426:Bacterial Proteins; D003373:Coumaric Acids; D020283:Photoreceptors, Microbial; D011422:Propionates; D011506:Proteins; D011993:Recombinant Fusion Proteins; C050162:photoactive yellow protein, Bacteria; C495469:p-coumaric acid
keywords: color tag; high-throughput protein expression screening; protein expression test; protein purification; protein purity estimation; protein quantification