Virtually all extracellular proteins in humans are glycoproteins and likewise are many biopharmaceuticals. The glycosylation is directly correlated to biological function and stability of these proteins. The ability to isolate glycoproteins is thus of great importance in many applications. The most common isolation method for glycoproteins is affinity chromatography using lectins, a ubiquitous and versatile group of carbohydrate-binding proteins. The lectin Concanavalin A (ConA) has long been used for this purpose but suffers from undesired leakage into the eluate, causing an inquiry of alternative chromatography ligands or optimization of the ConA resin.In this study, a total of 20 different lectins, including ConA, were evaluated and compared in terms of suitability as ligands in affinity chromatography for glycoprotein isolation. The lectins’ binding to glycoproteins were studied, mainly through microtiter plate binding assays using a monoclonal IgG1 antibody and Conalbumin (Ovotransferrin). Further, sugar-specificities and potential eluting sugars for the lectins were examined through inhibition with eight different carbohydrates. Additionally, the glycoprotein binding and leakage of ConA columns were examined, and a potential leakagereducing treatment of ConA resin evaluated.ConA was found to be superior in binding to the investigated glycoproteins but exhibited a limited binding when immobilized to an agarose resin. This discrepancy is likely a consequence of structurally hidden glycans on the used glycoproteins and requirements of long residence time when used in a chromatographic setting. Binding competition with several sugars were investigated with a similar microtiter plate binding assay. This method displayed potential to predict the behaviour of sugars and their suitability as eluting agents in a chromatography column. The best eluting sugar for ConA was showed to be methylmannoside, ideally in combination with methylglucoside. Lastly, evaluation of ConA columns with a crosslinking glutaraldehyde-treatment showed that the ConA ligand leakage may be significantly reduced, although further studies and optimizations are needed.This study thus presents a repertoire of lectins and their differences in terms of glycoprotein-binding and sugar-specificity, as well as evaluations of ConA columns’ efficiency and potential leakage-prevention.
| Identifer | oai:union.ndltd.org:UPSALLA1/oai:DiVA.org:uu-417024 |
| Date | January 2020 |
| Creators | Andersson, Pontus |
| Publisher | Uppsala universitet, Institutionen för biologisk grundutbildning |
| Source Sets | DiVA Archive at Upsalla University |
| Language | English |
| Detected Language | English |
| Type | Student thesis, info:eu-repo/semantics/bachelorThesis, text |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | UPTEC X ; 20008 |
Page generated in 0.0052 seconds