Targeted and proximity-dependent promiscuous protein biotinylation by a mutant Escherichia coli biotin protein ligase.
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.
Cronan
John E
JE
Department of Microbiology, University of Illinois, Urbana, IL 61801, USA. j-cronan@life.uiuc.edu
D000249:Adenosine Monophosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D001710:Biotin / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*; D019731:Carbon-Nitrogen Ligases / Q000235:genetics* / Q000378:metabolism*; D015500:Chloramphenicol O-Acetyltransferase / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism*; D007136:Immunoglobulins / Q000737:chemistry / Q000378:metabolism; D008239:Lysine / Q000378:metabolism; D009154:Mutation; D015202:Protein Engineering / Q000379:methods; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism*; D012259:Ribonuclease, Pancreatic / Q000737:chemistry / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism*
C082099:5'-AMP-biotin; D029968:Escherichia coli Proteins; D007136:Immunoglobulins; D012097:Repressor Proteins; D014157:Transcription Factors; D012710:Serum Albumin, Bovine; D000249:Adenosine Monophosphate; D001710:Biotin; D015500:Chloramphenicol O-Acetyltransferase; D012259:Ribonuclease, Pancreatic; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli; D008239:Lysine