New approaches to generating semi-synthetic post-translationally modified proteins

Genetic engineering has opened countless doors in biological research. However, while we have near complete control over the amino acid sequence of a protein of interest, the same degree of control has not been exerted over the many post-translational modifications that modulate protein functions in living cells. One of these modifications is the glycosylphosphatidylinositol (GPI) anchor. A difficulty inherent to studying GPI-anchored proteins is the lack of a heterologous expression system suited to expressing large quantities of these proteins. Recent developments in protein chemistry have enabled researchers to ligate synthetic and recombinant peptide segments together, and these segments have included chemically modified amino acids. The incorporation of proteins containing C-terminal GPI anchor analogs generated via chemical ligation reactions into cell membranes is presented. This thesis explores applications to production of GPI-modified proteins.; Another post-translational modification that scientists do not have complete control over is glycosylation, which can have a profound effect on protein folding and stability. The prion protein is an example of a pathological system in which glycosylation plays an important, yet to date, murky role in the protein's conformational properties. The role of glycans in prion disease strains and species barriers is not completely clear, but they seem to be important to phenotypic variations and transmission. These factors point to a need for the production of variably glycosylated prion protein for structural and functional studies. The attempts to generate a full-length, glycosylated prion protein through expressed protein ligation is presented.; Finally, I endeavored to expand the chemoselective ligation reactions available to protein chemists and provide new functional groups that could be appended to the C-termini of expressed proteins. The final chapter presents two new protein ligation reactions that enable modification via hydrazone chemistry.