A glycoprotein can exist as a spectrum of glycosylated forms, where each protein molecule may be associated with an array of oligosaccharide structures. The overall range of glycoforms can have a variety of different biophysical and biochemical properties. As a result of this 'microheterogeneity' there is a need for the synthesis of homogeneously glycosylated proteins for analytical purposes. The synthesis of novel glycoproteins through the selective reaction of glycosyl iodoacetamides with the thiol groups of cysteine has been developed (Figure 10919). Through site-directed mutagenesis, whereby the natural asparagine glycosylation sites can be exchanged for cysteine, it was possible to selectively glycosylated proteins at predetermined sites. This provided a general method for the synthesis of homogeneously glycosylated proteins. We chose the glycoprotein hormone erythropoietin as a model system since the <I>N</I>-glycans at residues 24, 38 and 83 are essential for <I>in vivo</I> biological activity. Using a modified recombinant erythropoietin gene (prepared in Chapter 2) we optimized protein expression, and over-expressed and purified His<SUB>10</SUB>-WThEPO, His<SUB>10</SUB>-Asn24Cys, His<SUB>10</SUB>-Asn38Cys, His<SUB>10</SUB>-Asn83Cys and His<SUB>10</SUB>-Asn38/83 CyshEPO's in yields of 13mgL<SUP>-1</SUP> from <I>E. coli.</I> This allowed us to probe the structure of rhEPO using techniques such as NMR spectroscopy and electrospray MS (Chapter 3). Having access to larger quantities of EPO, we were also able to develop on-line LC-ESI-MS methods, which allowed us to monitor protein glycosylation reactions with glycosyl β-<I>N</I>-iodoacetamides and map the position of the glycosylation site. The synthesis of relevant iodoacetamides was attempted and simple glycosyl iodoacetamides and <SUP>13</SUP>C labeled iodoacetamides were prepared and successfully employed as probes for protein glycosylation.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:657135 |
| Date | January 1999 |
| Creators | Macmillan, Derek |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/12523 |
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