Use of a promiscuous biotin ligase to identify partners of the CRTC transcription factor

• Main Author: Nai, Rui Si
• Other Authors: Mark Stephen Featherstone
• Format: Final Year Project
• Language: English
• Published: 2015
• Subjects: DRNTU::Science::Biological sciences::Genetics
• Online Access: http://hdl.handle.net/10356/65366
• Institution: Nanyang Technological University

CREB is an important transcription factor that is regulated in part by the CRTC family of cofactors. CRTCs work in tandem with CREB to mediate various metabolic processes, such as promoting gluconeogenesis in response to increased glucagon levels. CRTC1 in particular is highly expressed in the brain and known to be involved in memory formation, learning and synaptic plasticity. Consequently, its dysregulation is linked to neurological diseases as well as memory and learning problems. Identifying and studying novel CRTC1 partners may provide a better understanding of how CRTC1 functions. This project tests the feasibility of BioID in pulling down CRTC1 partner proteins. BioID is a protein screening technique that involves fusing a mutant E.coli biotin ligase, BirA*, to the protein of interest in order to biotinylate proximal proteins. These potential interaction partners can then be identified through streptavidin pull-down and Western blot or mass spectrometry. Results showed that known CRTC1 partners CREB and MEIS1A were successfully pulled down when overexpressed, demonstrating that BioID is functional and that novel protein partners of CRTC1 can potentially be pulled down with this method.