Affinity precipitation and macroaffinity ligand facilitated three-phase partitioning for refolding and simultaneous purification of urea-denatured pectinase.
Protein refolding is an integral step in the recovery of protein activity from inclusion bodies. It is shown that affinity precipitation and macroaffinity ligand facilitated three-phase partitioning (MLFTPP) led to refolding of urea-denatured pectinase present in a commercial preparation, with simultaneous purification. Affinity precipitation consists of precipitation of the desired enzyme by complexing it with a suitable stimulus-sensitive macroaffinity ligand. This ligand in this case was alginate/esterified alginate. The complex of the polymer-pectinase could be precipitated by adding calcium ions. In MLFTPP (carried out by adding tertiary butanol and ammonium sulfate to the aqueous solution of crude enzyme and the polymer), the polymer or its complex with the enzyme form an interfacial precipitate between tert-butyl alcohol phase and aqueous phase. It is believed that in both processes, while molecular recognition of alginate/esterified alginate to pectinase facilitates their selective binding to the enzyme, the correct refolding is facilitated by preventing molecular aggregation of unfolded enzyme molecules. Three-phase partitioning with esterified alginate as the macroaffinity ligand gave 100% recovery with 4-fold purification. Affinity precipitation with 1% alginate gave 52% yield with 18-fold purification. On the other hand, use of 0.5% esterified alginate gave only 7-fold purification but with 75% recovery of activity.
Chemistry Department, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi 110016, India.
Aparna
A
Sharma
Ipsita
I
Roy
Munishwar Nath
MN
Gupta
D000345:Affinity Labels; D011232:Chemical Precipitation; D004591:Electrophoresis, Polyacrylamide Gel; D008024:Ligands; D011096:Polygalacturonase / Q000737:chemistry / Q000302:isolation & purification*; D014508:Urea / Q000737:chemistry*
D000345:Affinity Labels; D008024:Ligands; D014508:Urea; D011096:Polygalacturonase