Selenolthiol and Dithiol C-Terminal Tetrapeptide Motifs for One-Step Purification and Labeling of Recombinant Proteins Produced in E. coli
✍ Scribed by Qing Cheng; Linda Johansson; Jan-Olov Thorell; Anna Fredriksson; Erik Samén; Sharon Stone-Elander; Elias S. J. Arnér
- Publisher
- John Wiley and Sons
- Year
- 2006
- Tongue
- English
- Weight
- 263 KB
- Volume
- 7
- Category
- Article
- ISSN
- 1439-4227
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✦ Synopsis
Abstract
We have previously shown that a redox‐active selenocysteine‐containing tetrapeptide—Sel‐tag (Gly‐Cys‐Sec‐Gly)—can be used as a C‐terminal fusion motif for recombinant proteins produced in Escherichia coli. This Sel‐tag allows selenolate‐targeted one‐step purification, as well as fluorescent labeling or radiolabeling either with gamma emitters (^75^Se) or with positron‐emitting radionuclides (^11^C). Here we have analyzed four different redox‐active C‐terminal motifs, carrying either dithiol (Gly‐Cys‐Cys‐Gly or Ser‐Cys‐Cys‐Ser) or selenolthiol (Gly‐Cys‐Sec‐Gly or Ser‐Cys‐Sec‐Ser) motifs. Utilizing these different functional motifs with the same recombinant protein (Fel d 1), we were able to assess their relative reactivities and potential usefulness for biotechnological applications. We found that all four redox‐active tags could be utilized for efficient one‐step purification to provide pure protein from a crude bacterial lysate through reversible binding to phenylarsine oxide sepharose, with yields and purities comparable to those obtained for a His‐tagged protein purified by the more common approach with use of a Ni^2+^ column. For labeling with electrophilic fluorescent or radioactive compounds, however, the selenolthiol motifs were considerably more efficient than their dithiol counterparts. The results thus show that both the selenolthiol‐ and the dithiol‐containing tags can serve as efficient alternatives to His‐tags for protein purification, while the selenolthiol motifs offer additional and unique potential for Sec‐targeted labeling. It should therefore be possible to utilize these multifunctional tetrapeptide motifs to develop a wide range of novel biotechnological applications based on Sec targeting with electrophilic compounds.