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High-throughput instant quantification of protein expression and purity based on photoactive yellow protein turn off/on label

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Title
High-throughput instant quantification of protein expression and purity based on photoactive yellow protein turn off/on label
Author(s)
Young Min Kim; Prabhakar Ganesan; Hyot Cherl Ihee
Publication Date
2013-08
Journal
PROTEIN SCIENCE, v.22, no.8, pp.1109 - 1117
Publisher
WILEY-BLACKWELL
Abstract
Quantifying the concentration and purity of a target protein is essential for highthroughput 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 coexpressed 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 highthroughput protein expression screening
URI
https://pr.ibs.re.kr/handle/8788114/1578
ISSN
0961-8368
Appears in Collections:
Center for Nanomaterials and Chemical Reactions(나노물질 및 화학반응 연구단) > Journal Papers (저널논문)
Files in This Item:
35_IBS CA1301-1_Protein science_22-01-1109-High-throughput instant quantification of protein expression and purity based on photoactive.pdfDownload

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