BirA enzyme: production and application in the study of membrane receptor-ligand interactions by site-specific biotinylation.
The enzyme BirA is a key reagent because of its ability to biotinylate proteins at a specific residue in a recognition sequence. We report a rapid, efficient, and economical method for the production, purification, and application of this enzyme. The method is easily scaled up and the protein produced is of high purity and can be stored for many months with retention of activity. We have used this enzyme to biotinylate the C termini of membrane proteins, allowing these proteins to be tetramerized by binding to streptavidin. Because of the specificity of the biotinylation at the C terminus, the orientation of the membrane proteins on the streptavidin is equivalent to that of the native protein on the cell surface. These tetrameric proteins can be used to study protein receptor-ligand interactions at the cell surface, and site-specific biotinylation can be used to study proteins in vitro using a defined orientation.
O'callaghan
C A
CA
Nuffield Department of Clinical Medicine, University of Oxford, Oxford, OX3 9DS, United Kingdom.
Byford
M F
MF
Wyer
J R
JR
Willcox
B E
BE
Jakobsen
B K
BK
McMichael
A J
AJ
Bell
J I
JI
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D001483:Base Sequence; D001671:Biochemistry / Q000379:methods*; D001709:Biotechnology / Q000379:methods; D001710:Biotin / Q000378:metabolism*; D019731:Carbon-Nitrogen Ligases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D029968:Escherichia coli Proteins; D015693:Gene Products, nef / Q000235:genetics / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006680:HLA Antigens / Q000235:genetics / Q000378:metabolism; D015395:Histocompatibility Antigens Class I / Q000378:metabolism; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D012097:Repressor Proteins; D020349:Surface Plasmon Resonance; D013601:T-Lymphocytes / Q000378:metabolism; D014157:Transcription Factors
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D015693:Gene Products, nef; D006680:HLA Antigens; D015395:Histocompatibility Antigens Class I; D011994:Recombinant Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D001710:Biotin; D000255:Adenosine Triphosphate; D005982:Glutathione Transferase; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli