Affinity-based separations take advantage of the high inherent selectivity afforded by biological molecules such as polysaccharides, nucleic acids, and proteins to purify molecules of interest. The proposed research involves merging the unique selectivity afforded by liquid-liquid extraction's using the newly developed technique -- ARMES (Affinity-based Reverse Micellar Extraction and Separation). The physicochemical factors that govern ARMES as well as a general mechanism of protein extraction into a surfactant-containing organic phase will be elucidated followed by the development of a quantitative understanding of the separation variables. Of primary importance to the proposed research is the use of lectins as affinity ligands in the ARMES approach to purify glycoproteins as well as glycoform variants. Many of the products prepared by biotechnological approaches, including recombinant genetic engineering, cell tissue culture, and monoclonal technologies, are glycoproteins. ARMES will be used to separate both positional glycoforms (e.g., different locations of sugar chain(s) on a glycoprotein) and heterogeneous glycoforms (e.g., different glycosylation chemistries at a given glycosylation site). Several models will be used for these glycoform separations including subtilisin BPN' neoglycoproteins (i.e., synthesized glycoproteins having well-defined properties), human kidney renin, and antithrombin III. The latter two are biomedically significant proteins. The successful isolation of these model glycoform variants will be useful also in the determination of the therapeutic efficacy of different glycoform variants of a biomedically important protein. The key features of the proposed research are: l. The elucidation of the physicochemical factors that govern ARMES; 2. The development of a quantitative understanding of the variables that affect protein extraction into organic solvents via the ARMES technique. 3. The evaluation of ARMES as a procedure to distinguish different glycoforms of glycoproteins using lectins as affinity ligands. 4. The investigation of ion-paired lectin-affinity interactions in organic solvents. 5. The identification of issues of importance for process optimization and economics.
| Boyer, L A; Shao, X; Ebright, R H et al. (2000) Roles of the histone H2A-H2B dimers and the (H3-H4)(2) tetramer in nucleosome remodeling by the SWI-SNF complex. J Biol Chem 275:11545-52 |
| Choe, J; Zhang, F; Wolff, M W et al. (2000) Separation of alpha-acid glycoprotein glycoforms using affinity-based reversed micellar extraction and separation. Biotechnol Bioeng 70:484-90 |
| Kuberan, B; Gunay, N S; Dordick, J S et al. (1999) Preparation and isolation of neoglycoconjugates using biotin-streptavidin complexes. Glycoconj J 16:271-81 |
| VanderNoot, V A; Hileman, R E; Dordick, J S et al. (1998) Affinity capillary electrophoresis employing immobilized glycosaminoglycan to resolve heparin-binding peptides. Electrophoresis 19:437-41 |
| Choe, J; VanderNoot, V A; Linhardt, R J et al. (1997) Parameters affecting the efficiency of affinity-based reversed micellar extraction and separation (ARMES) in glycoprotein purification. Biotechnol Prog 13:440-5 |
