Dual function labeling of biomolecules based on DsRed-monomer

A. M. Goulding, Y. Rahimi, S. Shrestha, S. K. Deo

Research output: Contribution to journalArticle

4 Scopus citations

Abstract

Unavailability of fusion tags that possess both affinity and visualization properties is a hurdle for biomolecular research. Typically, either a choice is made between an affinity tag and a reporter tag or both are employed in tandem if a fusion can be made at both termini of the target biomolecule. In this work, we have developed a site-specific genetic fusion approach employing DsRed-Monomer, a red fluorescent protein, that provides for both affinity and reporter functionality in a single tag. As a proof-of-concept, two fusion proteins, bradykinin-DsRed-Monomer and calmodulin-DsRed-Monomer, were prepared for the study. These fusion proteins were purified using a copper-immobilized column based on the inherent copper-binding property of DsRed-Monomer. Spectroscopic characterization of fusion proteins and comparison with native DsRed-Monomer showed no effect of fusion on the properties of DsRed-Monomer. Further, bradykinin-DsRed-Monomer was employed in the development of a competitive fluorescence immunoassay for the peptide bradykinin. Calmodulin-DsRed-Monomer was used to detect binding of the calmodulin ligand chlorpromazine, based on a change in the fluorescence of DsRed-Monomer upon binding of chlorpromazine to calmodulin. The studies performed demonstrate the application of DsRed-Monomer as a dual function tag indicating the potential usefulness of DsRed-monomer in proteomics and biomolecular research.

Original languageEnglish (US)
Pages (from-to)2113-2119
Number of pages7
JournalBioconjugate Chemistry
Volume19
Issue number11
DOIs
StatePublished - Nov 1 2008
Externally publishedYes

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biomedical Engineering
  • Pharmacology
  • Pharmaceutical Science
  • Organic Chemistry

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