Large Scale Peptide Synthesis and Purification

Cellular signalling cascades are the result of the transient modification of proteins with different post translational modifications (PTMs). In order to study the effect of these PTMs, they need to be introduced at specific sites on the protein backbone. This is challenging, as various post-translational modifications, including methylation, glycosylation, acetylation, ubiquitination, phosphorylation exist in continuous dynamic equilibrium and conventional expression can thus not provide these modified proteins. At present, only through chemical means can such distinctly and precisely modified proteins be obtained. To enable the required chemical synthesis, we seek funding to purchase a peptide synthesiser and a purification system capable of handling a range of coupling chemistries in large enough quantities to afford biochemical characterization and study. It will allow us to extend our expertise in synthesis of post translationally-modified (PTM)-peptides to those carrying multiple, different PTMs at specific sites on signalling protein backbones. The new synthesiser will enable production of small peptide libraries on a sufficiently large scale to allow the elucidation of biochemical properties of these different modiforms using techniques such as protein crystallography and NMR. Hereby we aim to create a new class of reagents for the study of these PTMs on the signalling of key cellular regulators, such as the tumour suppressor p53. This new synthesiser will also aid us in our existing projects on the synthesis of ubiquitinated proteins, single glycoforms of immune ligands, T-cell staining reagents and with the biochemical studies of deubiquitinating enzymes and ubiquitin binding domains. The peptide synthesiser will therefore not only be of great assistance to our existing projects, but will also allow the synthesis of a new class of reagents for studying cell signalling.