Lab rotation project description
The Notch proteins that span the cytoplasmic membrane are an evolutionarily conserved family of four different proteins at the head of cell-to-cell signalling cascades. These proteins have been shown to have an essential role in regulating development and homeostasis in many tissues.
They are involved in lineage commitment, cell cycle progression, differentiation and the maintenance and self-renewal of stem cells. Mutations in Notch pathway genes have been linked with many congenital disorders including Alagille, Hajdu-Cheney and Adam-Oliver syndromes as well as cerebral autosomal-dominant arteriopathy (CADASIL). Mutations have also been identified in a number of cancers (squamous cell carcinoma, small-cell carcinoma, B cell lymphoma).
A number of viral proteins from human herpes viruses, papillomavirus and Epstein-Barr viruses also target proteins in the Notch cascade. In this project, affibodies3 (small 58-residue, 6.5 kDa proteins derived from the Z domain triple helix of staphylococcal protein A) with a high affinity against the extracellular portion of a Notch protein will be selected from a phage display library. Affibodies are attractive alternatives to antibodies or their fragments as biological molecular recognition elements as they are considerably smaller than immunoglobulin G (150 kDa; 4 peptide chains linked by disulfides). Additionally, they do not contain cysteines, disulfide bonds or post-translational modifications allowing them to be expressed in a wide variety of hosts including E. coli. This makes them easy to engineer (randomization of 13 contiguous residues in the domain is well tolerated) and produce in large quantities. Affibodies with nM to pM affinities have been identified for EGFR, HER2 and interleukin receptors. The affibodies can also be easily modified with fluorophores or other imaging probes for use in vivo.
The project will involve the isolation of the extracellular portion of a Notch protein and its immobilization on magnetic beads. A phage display library of the affibody scaffold (M13 or T7 phage) will then be produced and panned against the Notch protein to select high affinity binders. After a number of rounds of panning, the highest affinity affibodies will be sequenced and re-expressed in soluble form for follow-on biological studies.