Investigating Oligosaccharyltransferases Of N-Linked Glycosylation Using Escherichia Coli.
Escherichia coli is a powerful tool for elucidating many of the basic principles of biology. As a protein production host, E. coli can produce exogenous protein to upwards of 20% of the total cellular protein content. While E. coli is used to produce a wide variety of proteins for research and therapeutic purposes, this organism is limited in its capability to perform various post-translational modifications required for the proper function of most mammalian proteins. One such modification is Nlinked glycosylation, the transfer of an oligosaccharide onto an asparagine residue within an acceptor peptide sequence. N-linked glycosylation can alter the solubility of a protein, enhance its effector function, and increase the serum half-life of therapeutic proteins. In recent years, researchers have reconstituted the N-linked glycosylation pathway from a distantly related gram-negative pathogen, Campylobacter jejuni, in non-pathogenic E. coli. While engineered strains of E. coli are now capable of Nlinked glycosylation, the type of glycan transferred, the acceptor protein sequence modified, and the efficiency of protein modification remain limiting factors in these recombinant systems. To address these limitations, this work focused on understanding and improving the oligosaccharyltransferase (OST), the central enzyme of the N-linked glycosylation pathway. First, selection tools were designed to isolate E. coli with enhanced glycosylation capabilities. One of these tools, the glycophage display system, was utilized to select for OSTs with altered acceptor site specificity. Next, we performed a functional analysis of twenty-three different bacterial OSTs. Harnessing the functional diversity in naturally occurring bacterial OSTs combined with structure-guided mutations yielded several interesting OSTs with unique acceptor site specificities not previously reported. Finally, to further simplify the model framework of glycosylation, we designed an in vitro prokaryotic-based cell-free system to efficiently synthesize glycoproteins. E. coli has proven to be a useful tool for developing a more thorough understanding of N-linked glycosylation, and continued research in the field of bacterial glycosylation will undoubtedly lead to new and important discoveries with significant impact on the biopharmaceutical industry.
Putnam, David A.; Quirk, Susan Mary; Oswald, Robert Edward
Ph.D. of Veterinary Medicine
Doctor of Philosophy
dissertation or thesis