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Site-specific modification of glycoproteins expressed in an engineered strain of Escherichia coli.

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Call No.	Ph.D. 2009
Author	Henderson, Grant E.
Title	Site-specific modification of glycoproteins expressed in an engineered strain of Escherichia coli.
Date	2009
Abstract	We have created an E. coli based platform for protein modification by coupling an engineered glycosylation pathway with a subsequent chemoenzymatic labeling strategy in which the glycans are oxidized to provide a bioorthogonal aldehyde group that can react with appropriate nucloephiles for the construction of bioconjugates. Both modes of glycosylation were studied. O-glycosylation, while successful in vitro, proved difficult to conduct in vivo. Several strategies were pursued in order to rectify this problem; however, solubility was rarely achieved and activity was nearly always absent. In contrast, the N-glycosylation pathway from C. jejuni readily yielded glycoproteins with appropriately reactive GalNAc terminated glycans. The glycan produced has the structure GalNAc-?1,4-GalNAc-?1,4-(Glc-?1,3-)GalNAc-?1,4-GalNAc-?1,4-GalNAc-?1,3-Bac. The presence of bacillosamine and multiple non-human linkages makes this glycan suboptimal for therapeutic use. Modification of the genetic locus responsible for glycocompetence in an effort to abrogate bacillosamine biosynthesis and limit GalNAc transfer resulted in a glycan of the composition Gal-HexNAc3. The terminal Gal was not expected and lack of reactivity in labeling reactions suggests that it may be present as galactofuranose. Further optimization for the removal of the terminal galactose residue has been initiated. Ultimately, it was shown that oxidation of the GalNAc containing glycoproteins produced in this work results in functionalized glycans that can be conjugated to molecules with carbonyl reactive functional groups such as EZ-Link-Biotin-PEG4-Hydrazide and aminooxy functionalized poly(ethylene glycol). The ability to produce site-specifically modified proteins in this manner should be a valuable tool for the development of novel bioconjugates.
Description	ix, 159 leaves : ill. (some col.); 28 cm
Format	application/pdf
Advisor	Tillman U Gerngross Karl Griswold, Ursula Gibson, Phillip Robbins, Huijuan Li, Natarajan Sethuraman
Subject	Glycoproteins -- Synthesis. Escherichia coli -- Biotechnology. Bioconjugates -- Synthesis. Protein engineering.
College	Thayer School of Engineering.
Issuing Body	Dartmouth Dissertations are made available through the Dartmouth Digital Library.
Degree	Thesis (Ph.D.)--Dartmouth College.
Copyright	All rights reserved. No part of this book may be reproduced in any form by any electronic or mechanical means without permission in writing from the author.
DOI	http://dx.doi.org/10.1349/ddlp.315

Title	Page 1
Format	application/pdf
Issuing Body	Dartmouth Dissertations are made available through the Dartmouth Digital Library.
Degree	Thesis (Ph.D.)--Dartmouth College.
Full Text	Site-Specific Modification of Glycoproteins Expressed in an Engineered Strain of E. coli A Thesis Submitted to the Faculty in partial fulfillment of the requirements for the degree of Doctor of Philosophy By GRANT E. HENDERSON Thayer School of Engineering Dartmouth College Hanover, New Hampshire August 2009 Examining Committee: Chairman: Prof. Tillman Gerngross Member: Prof. Karl Griswold Member: Prof. Ursula Gibson Member: Prof. Phil Robbins Member: Dr. Huijuan Li Member: Dr. Natarajan Sethuraman Brian W. Pogue Dean of Graduate Studies © 2009 Trustees of Dartmouth College Grant E. Henderson

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