Immobilized metal affinity chromatography without chelating ligands: purification of soybean trypsin inhibitor on zinc alginate beads.
Immobilized metal affinity chromatography (IMAC) is a widely used technique for bioseparation of proteins in general and recombinant proteins with polyhistidine fusion tags in particular. An expensive and critical step in this process is coupling of a chelating ligand to the chromatographic matrix. This chelating ligand coordinates metal ions such as Cu(2+), Zn(2+), and Ni(2+), which in turn bind proteins. The toxicity of chemicals required for coupling and their slow release during the separation process are of considerable concern. This is an important issue in the context of purification of proteins/enzymes which are used in food processing or pharmaceutical purposes. In this work, a simpler IMAC design is described which should lead to a paradigm shift in the application of IMAC in separation. It is shown that zinc alginate beads (formed by chelating alginate with Zn(2+) directly) can be used for IMAC. As "proof of concept", soybean trypsin inhibitor was purified 18-fold from its crude extract with 90% recovery of biological activity. The dynamic binding capacity of the packed bed was 3919 U mL(-1), as determined by frontal analysis. The media could be regenerated with 8 M urea and reused five times without any appreciable loss in its binding capacity.
Gupta
Munishwar N
MN
Chemistry Department, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi 110016, India. mn_gupta@hotmail.com
Jain
Sulakshana
S
Roy
Ipsita
I
D000464:Alginates; D002614:Chelating Agents / Q000633:toxicity; D002846:Chromatography, Affinity / Q000379:methods* / Q000592:standards; D004340:Drug Contamination / Q000517:prevention & control; D017743:Equipment Reuse; D005506:Food Contamination / Q000517:prevention & control; D020723:Glucuronic Acid; D006603:Hexuronic Acids; D008670:Metals / Q000737:chemistry; D010940:Plant Proteins / Q000302:isolation & purification*; D030262:Soybean Proteins / Q000302:isolation & purification*; D014361:Trypsin Inhibitors; D015032:Zinc; D000516:alpha-Amylases / Q000037:antagonists & inhibitors
D000464:Alginates; D002614:Chelating Agents; D006603:Hexuronic Acids; D008670:Metals; D010940:Plant Proteins; D030262:Soybean Proteins; D014361:Trypsin Inhibitors; D020723:Glucuronic Acid; D000516:alpha-Amylases; D015032:Zinc