Use of a biomimetic peptide in the design of a competitive binding assay for biotin and biotin analogues

Kalvin J. Gregory, Leonidas G. Bachas

Research output: Contribution to journalArticlepeer-review

7 Scopus citations

Abstract

A competitive binding assay for biotin, biocytin, and desthiobiotin utilizing a genetically engineered enzyme-ligand conjugate is described herein. This assay is unique in that the enzyme-ligand conjugate consists of the streptavidin binding peptide Strep-tag II, which mimics the binding of biotin to streptavidin, rather than biotin itself. This allows for the construction of a well-defined, oligosubstituted enzyme-ligand conjugate for which the site of attachment of the ligand on the enzyme is known precisely. The assay has detection limits of 5 × 10-8 M for biotin, 1 × 10-7 M for biocytin, and 2 × 10-6 M for desthiobiotin, and it serves as a model system in that it demonstrates the feasibility of using enzyme-ligand conjugates in which a peptide mimic of the analyte ligand is genetically fused to the enzyme. This avoids the problems associated with covalent attachment of the ligand to the enzyme, such as multiple substitution of the ligand and variability of the site of attachment. To our knowledge, this is the first example of using an enzyme-peptide mimic conjugate to detect a nonpeptide analyte.

Original languageEnglish (US)
Pages (from-to)82-88
Number of pages7
JournalAnalytical Biochemistry
Volume289
Issue number1
DOIs
StatePublished - Feb 1 2001
Externally publishedYes

Keywords

  • Biomimetic peptide
  • Biotin
  • Competitive binding assay
  • Strep-tag II

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Use of a biomimetic peptide in the design of a competitive binding assay for biotin and biotin analogues'. Together they form a unique fingerprint.

Cite this