Promiscuous protein biotinylation by Escherichia coli biotin protein ligase

Protein Sci. 2004 Nov;13(11):3043-50. doi: 10.1110/ps.04911804. Epub 2004 Sep 30.

Abstract

Biotin protein ligases (BPLs) are enzymes of extraordinary specificity. BirA, the BPL of Escherichia coli biotinylates only a single cellular protein. We report a mutant BirA that attaches biotin to a large number of cellular proteins in vivo and to bovine serum albumin, chloramphenicol acetyltransferase, immunoglobin heavy and light chains, and RNAse A in vitro. The mutant BirA also self biotinylates in vivo and in vitro. The wild type BirA protein is much less active in these reactions. The biotinylation reaction is proximity-dependent in that a greater extent of biotinylation was seen when the mutant ligase was coupled to the acceptor proteins than when the acceptors were free in solution. This approach may permit facile detection and recovery of interacting proteins by existing avidin/streptavidin technology.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Biotinylation*
  • Carbon-Nitrogen Ligases / genetics
  • Carbon-Nitrogen Ligases / metabolism
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism*
  • Humans
  • Mutation
  • Proteins / metabolism*
  • Repressor Proteins / genetics
  • Repressor Proteins / metabolism
  • Transcription Factors / genetics
  • Transcription Factors / metabolism

Substances

  • Escherichia coli Proteins
  • Proteins
  • Repressor Proteins
  • Transcription Factors
  • Carbon-Nitrogen Ligases
  • birA protein, E coli