Chung Jinhwa A, Wollack James W, Hovlid Marisa L, Okesli Ayse, Chen Yan, Mueller Joachim D, Distefano Mark D, Taton T Andrew
Department of Chemistry, University of Minnesota, Minneapolis, 55455, USA.
Anal Biochem. 2009 Mar 1;386(1):1-8. doi: 10.1016/j.ab.2008.09.007. Epub 2008 Sep 14.
Although protein prenylation is widely studied, there are few good methods for isolating prenylated proteins from their nonprenylated relatives. We report that crosslinked agarose (e.g., Sepharose) chromatography medium that has been chemically functionalized with beta-cyclodextrin (beta-CD) is extremely effective in affinity chromatography of prenylated proteins. In this study, a variety of proteins with C-terminal prenylation target ("CAAX box") sequences were enzymatically prenylated in vitro with natural and nonnatural prenyl diphosphate substrates. The prenylated protein products could then be isolated from starting materials by gravity chromatography or fast protein liquid chromatography (FPLC) on a beta-CD-Sepharose column. One particular prenylation reaction, farnesylation of an mCherry-CAAX fusion construct, was studied in detail. In this case, purified farnesylated product was unambiguously identified by electrospray mass spectrometry. In addition, when mCherry-CAAX was prenylated with a nonnatural, functional isoprenoid substrate, the functional group was maintained by chromatography on beta-CD-Sepharose, such that the resulting protein could be selectively bound at its C terminus to complementary functionality on a solid substrate. Finally, beta-CD-Sepharose FPLC was used to isolate prenylated mCherry-CAAX from crude HeLa cell lysate as a model for purifying prenylated proteins from cell extracts. We propose that this method could be generally useful to the community of researchers studying protein prenylation.
尽管蛋白质异戊二烯化已得到广泛研究，但从非异戊二烯化的相关蛋白中分离异戊二烯化蛋白的好方法却很少。我们报道，用β-环糊精（β-CD）进行化学功能化的交联琼脂糖（如琼脂糖凝胶）色谱介质在异戊二烯化蛋白的亲和色谱中极为有效。在本研究中，多种具有C端异戊二烯化靶点（“CAAX盒”）序列的蛋白在体外与天然和非天然异戊二烯二磷酸底物进行酶促异戊二烯化。然后，通过重力色谱或在β-CD-琼脂糖凝胶柱上的快速蛋白质液相色谱（FPLC），可以从起始材料中分离出异戊二烯化的蛋白产物。对一种特定的异戊二烯化反应，即mCherry-CAAX融合构建体的法尼基化进行了详细研究。在这种情况下，通过电喷雾质谱明确鉴定了纯化的法尼基化产物。此外，当mCherry-CAAX用非天然的功能性类异戊二烯底物进行异戊二烯化时，通过在β-CD-琼脂糖凝胶上的色谱法可保留该官能团，从而使所得蛋白的C端能够选择性地与固体底物上的互补官能团结合。最后，使用β-CD-琼脂糖凝胶FPLC从HeLa细胞粗裂解物中分离异戊二烯化的mCherry-CAAX，作为从细胞提取物中纯化异戊二烯化蛋白的模型。我们认为，该方法可能对研究蛋白质异戊二烯化的研究人员群体普遍有用。
