Directed Evolution of Streptavidin Variants Using In Vitro Compartmentalization

Matthew Levy, Andrew D. Ellington

Research output: Contribution to journalArticle

22 Scopus citations

Abstract

We have developed and implemented an in vitro compartmentalization (IVC) selection scheme for the identification of streptavidin (SA) variants with altered specificities for the biotin analog desthiobiotin. Wild-type SA and selected variants bind desthiobiotin with similar affinities (∼10-13 M), but the variants have off rates almost 50 times slower and a half-life for dissociation of 24 hr at 25°C. The utility of streptavidin variants with altered specificities and kinetic properties was shown by constructing protein microarrays that could be used to differentially organize and immobilize DNAs bearing these ligands. The methods we have developed should prove to be generally useful for generating a variety of novel SA reagents and for evolving other extremely high-affinity protein:ligand couples.

Original languageEnglish (US)
Pages (from-to)979-989
Number of pages11
JournalChemistry and Biology
Volume15
Issue number9
DOIs
StatePublished - Sep 22 2008

Keywords

  • CHEMBIO

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Pharmacology
  • Drug Discovery
  • Clinical Biochemistry

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