Selenolthiol and dithiol C-terminal tetrapeptide motifs for one-step purification and labeling of recombinant proteins produced in E. coli

Chembiochem. 2006 Dec;7(12):1976-81. doi: 10.1002/cbic.200600326.

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 (75Se) or with positron-emitting radionuclides (11C). 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 Ni2+ 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs*
  • Amino Acid Sequence
  • Base Sequence
  • Escherichia coli / chemistry
  • Escherichia coli / genetics*
  • Escherichia coli / metabolism
  • Fluorescent Dyes / chemistry*
  • Isotope Labeling
  • Molecular Sequence Data
  • Peptides / chemistry*
  • Recombinant Proteins / analysis*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Selenocysteine / chemistry*

Substances

  • Fluorescent Dyes
  • Peptides
  • Recombinant Proteins
  • Selenocysteine