Detection of Histidine-Tagged Protein in Escherichia coli by Single-Cell Inductively Coupled Plasma-Mass Spectrometry
2022
Tanaka, Yu-ki | Shimazaki, Shunsuke | Fukumoto, Yasunori | Ogra, Yasumitsu
We have developed a rapid and precise quantification method for a histidine (His)-tagged recombinant protein produced in Escherichia coli (E. coli) by single-cell inductively coupled plasma-mass spectrometry (SC-ICP-MS). Plasmid vector containing enhanced green fluorescent protein (EGFP) or red fluorescent protein (mCherry) gene fused with His-tag was transformed into E. coli. The transformed E. coli was exposed to nickel (Ni) chloride or cobalt (Co) chloride for labeling His-tag with the Ni or Co ion. Then, E. coli was analyzed by SC-ICP-MS to determine the amount of EGFP or mCherry protein on the basis of the signal of Ni or Co bound to His-tag. By comparing Ni and Co contents in E. coli expressing His-tagged mCherry with those in nontagged mCherry, the specific binding of Co to His-tag was more clearly detected than that of Ni. The Co contents were increased until 6 h after the protein induction, and this observation was coincident with the increases in fluorescence intensity of EGFP or mCherry measured by a flow cytometer. However, the Co contents were decreased for EGFP and kept at a constant level for mCherry from 6 to 24 h despite the continuous increase in the fluorescence intensity through incubation. The fluorescent proteins were mainly recovered in the insoluble fraction 24 h after the induction. This can be explained by the fact that the overexpressed fluorescent proteins with His-tag are transferred into inclusion bodies, which hampers the binding of the fluorescent proteins to the Co ion. SC-ICP-MS can be a useful technique to precisely quantify soluble recombinant proteins in E. coli without the extraction and purification process.
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