Synthesis of a radiolabeled zwitterionic detergent and its use in protein purification
Synthesis of a radiolabeled zwitterionic detergent and its use in protein purification
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DOI：
10.1006/abio.1998.2631
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发表时间：
1998-05-01
影响因子：
2.9
通讯作者：
Mellors, A
中科院分区：
文献类型：
--
作者：
Hannam, C;Lange, GL;Mellors, A
放射性标记允许检测残留在提取的疏水或膜蛋白中的痕量两性离子去污剂。为了开发其存在的灵敏度和特异性测定，以氚化形式合成了常用的两性离子膜蛋白去污剂3-[（3-胆酰胺丙基）二甲基铵基]1-丙磺酸盐（Chaps）。通过7-酮脱氧胆酸合成得到[7-H-3]Chaps，产率为53%，比活性为0.85 mCi/mmol。一种新的胆酸溶剂萃取系统，避免了这种中间体的色谱分离的需要。该方案可以容易地修改以产生更高比活性的[7-H-3]Chaps。[7-H-3]Chaps用于监测从含有0.2%（w/v）Chaps的浓缩细菌培养物上清液中去除去污剂的各种策略的效率。透析去除了95%的Chaps，并且向透析缓冲液中加入洗涤剂亲和珠导致97%的Chaps去除。凝胶过滤层析去除了99.9%的Chaps，而去污剂亲和珠层析柱去除了99.99%。总体而言，凝胶过滤色谱法是最方便和最经济的方法，从复杂的生物混合物中一步去除两性剂。(C)北京：科学出版社.
Radiolabeling permits the detection of trace amounts of zwitterionic detergent remaining in extracted hydrophobic or membrane proteins. To develop a sensitive and specific assay for its presence, the commonly used zwitterionic membrane protein detergent 3-[ (3-cholamidopropyl) dimethylammonio]1-propanesulfonate (Chaps) was synthesized in a tritiated form. Synthesis via 7-ketodeoxycholic acid gave [7-H-3]Chaps in 53% yield with a specific activity of 0.85 mCi/mmol. A novel solvent extraction system for cholic acid obviated the need for chromatographic isolation of this intermediate. The protocol can be readily modified to yield [7-H-3]Chaps of higher specific activity. [7-H-3]Chaps was used to monitor the efficiency of various strategies for detergent removal from concentrated bacterial culture supernatants containing 0.2% (w/v) Chaps. Dialysis removed 95% of Chaps and the addition of detergent-affinity beads to the dialysis buffer resulted in 97% removal of Chaps. Gel-filtration chromatography removed 99.9% of Chaps, while a detergent-affinity bead chromatography column removed 99.99%. Overall, gel-filtration chromatography was the most convenient and economical method for the one-step removal of the zwittergent from complex biological mixtures. (C) 1998 Academic Press.