Maximizing Yield and Modification of 13C/15N Labeled Heparosan From E. coli K5 Culture
Description
Understanding glycosaminoglycans’ (GAG) interaction with proteins is of growing interest for therapeutic applications. For instance, heparin is a GAG exploited for its ability to inhibit proteases, therefore inducing anticoagulation. For this reason, heparin is extracted in mass quantities from porcine intestine in the pharmaceutical field. Following a contamination in 2008, alternative sources for heparin are desired. In response, much research has been invested in the extraction of the naturally occurring polysaccharide, heparosan, from Escherichia coli K5 strain. As heparosan contains the same structural backbone as heparin, modifications can be made to produce heparin or heparin-like molecules from this source. Furthermore, isotopically labeled batches of heparosan can be produced to aid in protein-GAG interaction studies. In this study, a comparative look between extraction and purification methods of heparosan was taken. Fed-batch fermentation of this E. coli strain followed by subsequent purification yielded a final 13C/15N labeled batch of 90mg/L of heparosan which was then N-sulfated. Furthermore, a labeled sulfated disaccharide from this batch was utilized in a protein interaction study with CCL5. With NMR analysis, it was found that this heparin-like molecule interacted with CCL5 when its glucosamine residue was in a β-conformation. This represents an interaction reliant on a specific anomericity of this GAG molecule.
Date Created
The date the item was original created (prior to any relationship with the ASU Digital Repositories.)
2015-05
Agent
- Author (aut): Hoffman, Kristin Michelle
- Thesis director: Wang, Xu
- Committee member: Cabirac, Gary
- Committee member: Morgan, Ashli
- Contributor (ctb): Barrett, The Honors College
- Contributor (ctb): School of International Letters and Cultures
- Contributor (ctb): School of Life Sciences