Carboxymethyl cellulose as a new heterobifunctional ligand carrier for affinity precipitation of proteins.
Affinity precipitation is a technique that imparts selectivity to the widely used primary purification step of precipitation of proteins from crude extracts. Hetero-bifunctional affinity precipitation involves use of reversibly soluble/insoluble polymers that can be used as backbones to conjugate affinity ligands for specific separations. A variety of such polymers have been reported in literature. In this work we report development of carboxymethyl cellulose (CM cellulose) as a cheap, readily available and versatile reversibly soluble polymer system. Available CM cellulose as sodium salt could be quantitatively precipitated from its aqueous solution in presence of about 50 mM calcium and 7.2% w/v polyethylene glycol-4000, and could be resolubilised in the working buffer in absence of calcium, polyethylene glycol or both. Effectiveness of the CM cellulose-calcium-polyethylene glycol system was demonstrated by purifying lactate dehydrogenase from porcine muscle extract using covalently conjugated Cibacron blue dye-ligand. By careful choice of conditions that suppressed non-specific interactions, the system was shown to be an effective affinity precipitation polymer system inspite of the polyelectrolytic nature of CM cellulose. Up to 23 fold purification of the enzyme from crude extarct was obtained in one single precipitation sequence.
Lali
A
A
Department of Chemical Technology, University of Mumbai, India.
Balan
S
S
John
R
R
D'Souza
F
F
D000818:Animals; D002266:Carboxymethylcellulose Sodium / Q000378:metabolism*; D011232:Chemical Precipitation; D002846:Chromatography, Affinity / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D009132:Muscles / Q000201:enzymology; D009994:Osmolar Concentration; D011485:Protein Binding; D013552:Swine
D008024:Ligands; D002266:Carboxymethylcellulose Sodium