RESUMO
Due to its strength and specificity, the interaction between avidin and biotin has been used in a variety of medical and scientific applications ranging from drug targeting to immunohistochemistry. To maximize the application of this technology in mammalian systems, we recently demonstrated the ability to metabolically biotinylate tagged proteins in mammalian cells using the endogenous biotin ligase enzymes of the mammalian cell. This technology allows site-specific biotinylation without any exogenous reagents and eliminates possible inactivation of the protein of interest by nonspecific biotinylation. Here, we report further expansion of the mammalian metabolic biotinylation technology to enable biotinylation of proteins secreted from mammalian cells and expressed on their cell surface by cosecretion with BirA, the biotin ligase of E. coli. This technique can be used to biotinylate secreted proteins for purification or targeting and also for biotinylating the surfaces of mammalian cells to facilitate their labeling and purification from other nontagged cells.
Assuntos
Biotina/metabolismo , Proteínas de Escherichia coli , Proteínas de Membrana/metabolismo , Proteínas Repressoras , Fatores de Transcrição , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biotinilação/métodos , Western Blotting , Células CHO , Carbono-Nitrogênio Ligases/genética , Carbono-Nitrogênio Ligases/metabolismo , Carboxil e Carbamoil Transferases/genética , Carboxil e Carbamoil Transferases/metabolismo , Cricetinae , Eletroforese em Gel de Poliacrilamida , Escherichia coli/enzimologia , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Proteínas Luminescentes/isolamento & purificação , Proteínas Luminescentes/metabolismo , Proteínas de Membrana/genética , Propionibacterium/enzimologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
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.
