Discriminatory protein binding by a library of 96 new affinity resins: a novel dye-affinity chromatography tool-kit.
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.
Kumar
Sunil
S
Biotechnology Group, Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Powai, Mumbai 400 076, India.
Dalvi
Darshan B
DB
Moorthy
Masana
M
Korde
Shilpa S
SS
Fondekar
Kamalesh Pai
KP
Sahasrabudhe
Suhas D
SD
Schacht
Hans-Thomas
HT
Ekkundi
Vadiraj S
VS
Halik
Christine
C
Choudhury
Rajarshi
R
Kumar
Awanit
A
Punekar
Narayan S
NS
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
D007074:Immunoglobulin G; D012709:Serum Albumin; D014227:Triazines; C012842:Cibacron Blue F 3GA