title: Affinity binding of inclusion bodies on supermacroporous monolithic cryogels using labeling with specific antibodies.
abstract: A new chromatographic method based on affinity supermacroporous monolithic cryogels is developed for binding and analyzing inclusion bodies during fermentation. The work demonstrated that it is possible to bind specific IgG and IgY antibodies to the 15 and 17 amino acids at the terminus ends of a 33 kDa target protein aggregated as inclusion bodies. The antibody treated inclusion bodies from lysed fermentation broth can be specifically retained in protein A and pseudo-biospecific ligand sulfamethazine modified supermacroporous cryogels. The degree of binding of IgG and IgY treated inclusion bodies to the Protein A and sulfamethazine gels are investigated, as well as the influence of pH on the sulfamethazine ligand. Optimum binding of 78 and 72% was observed on both protein A and sulfamethazine modified cryogel columns, respectively, using IgG labeling of the inclusion bodies. The antibody treated inclusion bodies pass through unretained in the sulfamethazine supermacroporous gel at pH that does not favour the binding between the ligand on the gel and the antibodies on the surface of inclusion bodies. Also the unlabeled inclusion bodies went through the gel unretained, showing no non-specific binding or trapping within the gel. These findings may very well be the foundation for the building of a powerful analytical tool during fermentation of inclusion bodies as well as a convenient way to purify them from fermentation broth. These results also support our earlier findings [Kumar, A., Plieva, F.M., Galaev, I.Yu., Mattiasson, B., 2003. Affinity fractionation of lymphocytes using a monolithic cyogel. J. Immunol. Methods 283, 185-194] with mammalian cells that were surface labeled with specific antibodies and recognized on protein A supermacroporous gels. A general binding and separation system can be established on antibody binding cryogel affinity matrices.
authors.affiliation: Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-22100 Lund, Sweden.
authors.forename: Josefin
authors.initials: J
authors.lastname: Ahlqvist
authors.forename: Ashok
authors.initials: A
authors.lastname: Kumar
authors.forename: Heléne
authors.initials: H
authors.lastname: Sundström
authors.forename: Erika
authors.initials: E
authors.lastname: Ledung
authors.forename: E Gunnar
authors.initials: EG
authors.lastname: Hörnsten
authors.forename: Sven-Olof
authors.initials: SO
authors.lastname: Enfors
authors.forename: Bo
authors.initials: B
authors.lastname: Mattiasson
mesh_terms: D000906:Antibodies / Q000737:chemistry*; D002458:Cell Fractionation / Q000379:methods*; D002846:Chromatography, Affinity / Q000379:methods*; D005782:Gels / Q000737:chemistry; D007074:Immunoglobulin G / Q000737:chemistry; D007136:Immunoglobulins / Q000737:chemistry; D002479:Inclusion Bodies / Q000737:chemistry* / Q000276:immunology; D016062:Porosity; D011506:Proteins / Q000302:isolation & purification*; D013205:Staphylococcal Protein A / Q000737:chemistry; D013418:Sulfamethazine / Q000737:chemistry
chemical_list: D000906:Antibodies; D005782:Gels; C016512:IgY; D007074:Immunoglobulin G; D007136:Immunoglobulins; D011506:Proteins; D013205:Staphylococcal Protein A; D013418:Sulfamethazine