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FOR THE RECORD

Promiscuous protein biotinylation by Escherichia coli biotin protein ligase

¹ Departments of Microbiology and ² Biochemistry, University of Illinois, Urbana, Illinois 61801, USA
³ SurroMed, Inc., Menlo Park, California 94025, USA

(RECEIVED June 7, 2004; FINAL REVISION July 15, 2004; ACCEPTED July 15, 2004)

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.

Keywords: biotin protein ligase; protein modification; biotinylation; acyl adenylate; BirA

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04911804.

Reprint requests to: John E. Cronan, Departments of Microbiology and Biochemistry, University of Illinois, Urbana, IL 61801, USA; e-mail: j-cronan{at}life.uiuc.edu; fax: (217) 244-6697.

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