Skip to main content

Protein complex purification by in vivo biotinylation

Final Activity Report Summary - BIOTINYLATION (Protein complex purification by in vivo biotinylation)

Novel methodology involving the specific in vivo biotinylation of tagged proteins in mammalian cells has been developed. This project was focused on the application and establishment of this methodology in the host laboratory. Under this scheme a bio-tag was fused to specific proteins and was co-expressed in mammalian cells with a bacterial biotin transferase, which recognises and specifically biotinylates the tagged protein. The biotin by virtue of its binding to avidin, provides a very high affinity tag for the purification of multiprotein complexes associated with the biotinylated tagged protein. Complexes of biotinylated proteins are purified by binding to streptavidin and bound proteins are eluted and identified by mass spectrometry.

This methodology can be also applied for identification of target genes of a specific DNA bound factor of interest. During this project the in vivo biotinylation method was applied for one step high affinity approach in purifying novel protein complexes of proteins/factors involved in leukemias/cancer. Basic molecular mechanisms causing leukemias/cancer will be elucidated through this project and our future goal will be to translate this knowledge into new strategies for clinical management, at the preventive, diagnostic and therapeutic level.