Metabolic biotinylation of recombinant proteins in mammalian cells and in mice.
The avidin-biotin system is a fundamental technology in biomedicine for immunolocalization, imaging, nucleic acid blotting, and protein labeling. While this technology is robust, it is limited by the fact that mammalian proteins must be expressed and purified prior to chemical biotinylation using cross-linking agents which modify proteins at random locations to heterogeneous levels and can inactivate protein function. To circumvent this limitation, we demonstrate the ability to metabolically biotinylate tagged proteins in mammalian cells and in mice using the endogenous biotinylation enzymes of the host. Endogenously biotinylated proteins were readily purified from mammalian cells using monomeric avidin and eluted under nondenaturing conditions using only biotin as the releasing agent. This technology should allow recombinant proteins and fragile protein complexes to be produced and purified from mammalian cells as well as from transgenic plants and animals. In addition, this technology may be particularly useful for cell-targeting applications in which proteins or viral gene therapy vectors can be biotinylated at genetically defined sites for combination with other targeting moieties complexed with avidin.
Parrott
M B
MB
Department of Microbiology and Immunology, Baylor College of Medicine, Houston, Texas 77030, USA.
Barry
M A
MA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001710:Biotin / Q000378:metabolism*; D016466:CHO Cells; D019878:Carboxyl and Carbamoyl Transferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002460:Cell Line; D006224:Cricetinae; D017931:DNA Primers / Q000235:genetics; D005260:Female; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008722:Methods; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011424:Propionibacterium / Q000201:enzymology / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D012867:Skin / Q000378:metabolism; D014162:Transfection
D017931:DNA Primers; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D001710:Biotin; D019878:Carboxyl and Carbamoyl Transferases; C021580:Methylmalonyl-CoA carboxytransferase