Biotination of proteins in vivo. A post-translational modification to label, purify, and study proteins.
Post-translational modification of proteins with biotin provides the means to specifically label proteins in vivo and to purify proteins from crude cell lysates. The carboxyl-terminal protein segments modified by reaction with biotin ligase are strongly conserved in nature. We have demonstrated that the proteins encoded by translational gene fusions of a number of heterologous proteins to these carboxyl-terminal sequences become biotinated in vivo. The minimum size of the protein segment needed to allow biotination of fusion proteins is 75 amino acids. This biotination sequence, although of bacterial origin, functions in Saccharomyces cerevisiae as well as in Escherichia coli. Fusion proteins are readily labeled with [3H]biotin in vivo and the labeling is highly specific due to the scarcity (less than 5) of biotinated protein species. Biotinated fusion proteins can be readily purified in native form by binding to columns of monomeric avidin followed by elution with buffers containing biotin. Alternatively, proteins can be purified in a denatured form in presence of 1% sodium dodecyl sulfate or 8 M urea. Thus, this technology allows purification by affinity chromatography of any protein to which a biotination sequence can be attached. The ability to specifically label a protein in vivo should have utility in studies such as intracellular protein trafficking and cytoskeletonal dynamics.
Cronan
J E
JE
Department of Microbiology, University of Illinois, Urbana 61801.
D000595:Amino Acid Sequence; D001710:Biotin / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005696:Galactosidases / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D001616:beta-Galactosidase / Q000235:genetics* / Q000302:isolation & purification
D011506:Proteins; D011993:Recombinant Fusion Proteins; D001710:Biotin; D005696:Galactosidases; D001616:beta-Galactosidase