ENGINEERING WATER-SOLUBLE VARIANTS OF THE SINGLE-SUBUNIT OLIGOSACCHARYLTRANSFERASE
Kwon, Yong Hyun
Oligosaccharyltransferase (OST) is a key enzyme in the asparagine-linked (N-linked) protein glycosylation pathway. OSTs exist in all domains of life and are capable of transferring a preassembled glycan from lipid carrier to an acceptor peptide. Bacterial OSTs are an single-subunit enzyme that are amenable to recombinant expression in model organism including Escherichia coli. As a result, bacterial OSTs have been used as models to explore the mechanism of the N-linked glycosylation process in nature. These developments, notwithstanding, recombinant expression and purification of the OST enzymes remain significant challenges. Bacterial OSTs are multi-pass transmembrane protein that requires intricate balance between protein synthesis rate and a pace of membrane insertion. Further, membrane protein purification often necessitates the use of ultracentrifugation and detergent, both of which limit process scalability and compatibility. To address these challenges, we proposed a protein engineering strategy called SIMPLEx or solubilization of integral membrane proteins with high levels of expression to generate water-soluble variants of the bacterial OST. Specifically, we designed several OST chimeras where the N-terminus of the OST is fused with the amphipathic protein including engineered human apolipoprotein A-I. Using E. coli culture as an expression platform, several SIMPLEx-OSTs could be expressed within the cytoplasmic fraction of the E. coli. Importantly, our engineered OSTs retain their biological activity and are able to N-glycosylate several acceptor proteins including therapeutic human growth hormone. Collectively, our OST-engineering strategy is anticipated to generate a new subclass of water-soluble N-OST enzymes with applications in bioproduction of the glycotherapeutics and glycovaccines.
Glyco; membrane; Oligosaccharyltransferase; PglB
M.S., Chemical Engineering
Master of Science
dissertation or thesis