Targeted and proximity-dependent promiscuous protein biotinylation by a mutant Escherichia coli biotin protein ligase☆
Received 28 March 2005; received in revised form 28 March 2005; accepted 28 March 2005.
Abstract
A method for general protein biotinylation by enzymatic means has been developed. A mutant form (R118G) of the biotin protein ligase (BirA) of Escherichia coli is used and biotinylation is thought to proceed by chemical acylation of protein lysine side chains by biotinoyl-5′-AMP released from the mutant protein. Bovine serum albumin, chloramphenicol acetyltransferase, immunoglobulin chains and RNAse A as well as a large number of E. coli proteins have been biotinylated. The biotinylation reaction is proximity dependent in that the extent of biotinylation is much greater when the ligase is coupled to the acceptor protein than when the acceptor is free in solution. This is presumably due to rapid hydrolysis of the acylation agent, biotinoyl-5′-AMP. Therefore, when the mutant ligase is attached to one partner involved in a protein–protein interaction, it can be used to specifically tag the other partner with biotin, thereby permitting facile detection and recovery of the proteins by existing avidin/streptavidin technology.
Department of Microbiology, University of Illinois, Urbana, IL 61801, USA
Department of Biochemistry, University of Illinois, Urbana, IL 61801, USA
Department of Microbiology, University of Illinois, Urbana, IL 61801, USA. Tel.: +1 217 333 7919; fax: +1 217 244 6697.
☆ This paper was presented at the international symposium “Vitamins as Regulators of Genetic Expression: Biotin as a Model” NAFTA Satellite Meeting to the XXV National Congress of Biochemistry held on December 3–4, 2004, in Ixtapa, Zihuatanejo, Mexico. This meeting was sponsored by Sociedad Mexicana de Bioquimica A.C.; Programa de Doctorado en Ciencias Biomedicas, Universidad Nacional Autonoma de Mexico; Laboratorios Roche–Syntex, Mexico; and Instituto de Investigaciones Biomedicas, Universidad Nacional Autonoma de Mexico.
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