title: Discriminatory protein binding by a library of 96 new affinity resins: a novel dye-affinity chromatography tool-kit.
abstract: Initial acceptance of Cibacron Blue 3G-A based matrices has made dye-ligand affinity chromatography an attractive proposition. This prompted the synthesis and search for new dye structures. A systematic library of 96 affinity resins was generated using novel analogs of Cibacron Blue 3G-A and also by varying spacer lengths for immobilization. The library was tested in a batch binding and elution mode using seven different proteins--four Aspergillus enzymes namely, NADP-glutamate dehydrogenase, laccase, glutamine synthetase and arginase, bovine pancreatic trypsin and the two serum proteins human serum albumin and immunoglobulin G. Unique binding patterns were observed for each of them indicating that the library displayed discriminatory interactions. The significance of spacer length in the interaction with proteins was discernable. Trypsin interacted best with affinity resins that had no spacer. It was possible to resolve IgG and HSA from a mixture using a combination of resins. There was a good spread of HSA binding capacity in the 96 affinity resins. While some showed better HSA binding capacity than the commercial CB3GA-based matrix, a few with lower capacity were also observed. Subsequent to an initial screen, one affinity resin (CR-017) could be used to enrich Aspergillus terreus NADP-GDH from crude cell extracts. The efficacy of this dye-affinity resin was rationalized by characterizing NADP-GDH inhibition kinetics with the corresponding free dye ligand. In the sum, the library provides a set of dye-ligand affinity matrices with a potential for use in high throughput screening for protein purification.
authors.affiliation: Biotechnology Group, Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Powai, Mumbai 400 076, India.
authors.forename: Sunil
authors.initials: S
authors.lastname: Kumar
authors.forename: Darshan B
authors.initials: DB
authors.lastname: Dalvi
authors.forename: Masana
authors.initials: M
authors.lastname: Moorthy
authors.forename: Shilpa S
authors.initials: SS
authors.lastname: Korde
authors.forename: Kamalesh Pai
authors.initials: KP
authors.lastname: Fondekar
authors.forename: Suhas D
authors.initials: SD
authors.lastname: Sahasrabudhe
authors.forename: Hans-Thomas
authors.initials: HT
authors.lastname: Schacht
authors.forename: Vadiraj S
authors.initials: VS
authors.lastname: Ekkundi
authors.forename: Christine
authors.initials: C
authors.lastname: Halik
authors.forename: Rajarshi
authors.initials: R
authors.lastname: Choudhury
authors.forename: Awanit
authors.initials: A
authors.lastname: Kumar
authors.forename: Narayan S
authors.initials: NS
authors.lastname: Punekar
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods*; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry; D008962:Models, Theoretical; D011485:Protein Binding; D012709:Serum Albumin / Q000737:chemistry; D014227:Triazines / Q000737:chemistry
chemical_list: D007074:Immunoglobulin G; D012709:Serum Albumin; D014227:Triazines; C012842:Cibacron Blue F 3GA