CA2+-INDUCED HYDROPHOBIC SITE ON CALMODULIN - APPLICATION FOR PURIFICATION OF CALMODULIN BY PHENYL-SEPHAROSE AFFINITY-CHROMATOGRAPHY
CA2+-INDUCED HYDROPHOBIC SITE ON CALMODULIN - APPLICATION FOR PURIFICATION OF CALMODULIN BY PHENYL-SEPHAROSE AFFINITY-CHROMATOGRAPHY
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DOI：
10.1016/0006-291x(82)90712-4
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发表时间：
1982-01-01
影响因子：
3.1
通讯作者：
ANDERSON, WB
中科院分区：
文献类型：
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作者：
GOPALAKRISHNA, R;ANDERSON, WB
钙调素在微摩尔浓度的Ca2+存在下定量地与苯基- sepharose和辛基- sepharose亲和柱结合。除了EGTA[乙二醇双β]。钙调素也可以用低离子强度缓冲液、非离子洗涤剂（即1% Triton X-100）或乙二醇（50%）从这些亲和柱中洗脱出来，这表明它们之间存在疏水相互作用。利用疏水相互作用色谱法，可以一步纯化牛脑匀浆中的钙调蛋白。为了大规模纯化，在应用于亲和柱之前，用等电沉淀法浓缩含有钙调素的蛋白质部分。该方法获得的产率（160-180 mg钙调素/kg脑）明显更高，所需时间（约为1 / 3）。5h)比先前描述的钙调素纯化过程要少得多。在钙调素亲和纯化方面，苯基- sepharose明显优于吩噻嗪- sepharose。
Calmodulin binds quantitatively to phenyl-Sepharose and octyl-Sepharose affinity columns in the presence of micromolar concentrations of Ca2+. In addition to EGTA [ethylene glycol bis(.beta.-aminoethyl ether)-N,N,N'',N''-tetraacetic acid], calmodulin also can be eluted from these affinity columns with low ionic strength buffer, non-ionic detergent (i.e., 1% Triton X-100) or ethylene glycol (50%), suggesting hydrophobic interaction. Using hydrophobic interaction chromatography calmodulin can be purified to homogeneity from bovine brain homogenate in a single step. For large-scale purification the protein fraction containing calmodulin was concentrated by isoelectric precipitation prior to application to the affinity column. The yield obtained by this procedure (160-180 mg calmodulin/kg brain) is significantly greater, and the time required (.apprx. 5 h) is substantially less, than that of previously described procedures for calmodulin purification. It is apparent that phenyl-Sepharose offers several advantages over phenothiazine-Sepharose for affinity purification of calmodulin.