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Effect of divalent ions on protein precipitation with polyethylene glycol: Mechanism of action and applications

1991; Elsevier BV; Volume: 2; Issue: 1 Linguagem: Inglês

10.1016/1046-5928(91)90015-b

1096-0279

Sonja L. Thrash, James Otto, Thomas L. Deits,

Protein purification and stability

Polyethylene glycol (PEG) is extensively employed for protein purification by fractional precipitation. Efficiency of precipitation is highest when the solution pH is near the isoelectric point of the target protein. At pH values far from the isoelectric point of the target protein, proteins develop a net positive or negative charge and are more resistant to precipitation. We have found that divalent cations (Ba2+, Sr2+, and Ca2+) or divalent anions (SO42−) significantly change the pattern of PEG precipitation when the ion is chosen so as to counteract the expected net charge on the target protein. At moderate (5–50 mm) concentrations of Ba2+, negatively charged proteins can be precipitated from solution at pH values as high as 10 with efficiency unchanged from precipitation at pH values near their isoelectric point values. The mechanism of PEG precipitation of protein at these high pH values appears to be unchanged from the mechanism operative at the protein isoelectric point. Precipitation is rapid and the capacity for protein precipitation is high. There is no detectable coprecipitation of small molecules (AMP, ATP, and NADH) or soluble proteins (carbonic anhydrase) induced when large quantities of protein are precipitated by this method. The purification of bovine carbonic anhydrase from erythrocyte lysate is more efficient at pH 10 in the presence of Ba2+ than is conventional PEG precipitation carried out at the isoelectric point of carbonic anhydrase. Application of these observations should broaden the utility of protein purification by fractional precipitation with PEG.