BIOTINYLATION · Protein complex purification by in vivo biotinylation
FP6 — Marie Curie Actions (Human Resources and Mobility)
- Duration
- 2005-05-01 → 2006-04-30
- EU contribution
- €40,000
- Participants
- 1
- Scheme
- ERG
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Results in brief
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.
Data: CORDIS, © European Union
Project objective
Novel methodology involving the specific in vivo biotinylation of tagged proteins in mammalian cells has been developed. The proposed project entails the application of this methodology as a one step high affinity approach in purifying novel protein complexes of the hybrid proteins formed from translocations in Chronic MyeloProliferative Disorders (CMPDs).The CMPDs, a category of leukaemias, are characterized by uncontrolled proliferation of one or several cell types of the myeloid or haematopoietic lineages. Translocations found in CMPDs create hybrid genes, producing hybrid proteins functioning as activated tyrosine kinases that trigger the uncontrolled cell proliferation. The proposed project will focus on the purification of complexes of two such hybrid proteins: BCR-FGFR1 and BCR-PDGFRA.A bio-tag will be fused to the specific proteins and will be co-expressed in mammalian cells with a bacterial biotin transferase, which recognises and specifically biotinylates the tagged protein. Complexes of biotinylated proteins will be purified by binding to streptavidin and bound proteins will be eluted and identified by mass spectrometry. This project will elucidate the molecular mechanisms causing the CMPDs and will provide insights in the field of proteomics for these disorders.The future goal will be to translate this knowledge into new strategies for clinical management, at both the diagnostic and therapeutic level (i.e. identification of novel inhibitors of the proteins identified for treatment of CMPDs and potential siRNAs assays to inhibit the hybrid proteins or their partners).
Original text from CORDIS.
Participants
- FOUNDATION FOR BIOMEDICAL RESEARCH OF THE ACADEMY OF ATHENS · ATHENSCoordinatorCity levelGreece
Links
Data: CORDIS, © European Union