Farnesylation or geranylgeranylation? Efficient assays for testing protein prenylation in vitro and in vivo.
Farnesylation or geranylgeranylation? Efficient assays for testing protein prenylation in vitro and in vivo.
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法尼化或黄烷基凝聚力？在体外和体内测试蛋白质原始化的有效测定。
DOI：
10.1186/1471-2091-7-6
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发表时间：
2006-02-28
期刊：
影响因子：
--
通讯作者：
Eisenhaber, Frank
中科院分区：
文献类型：
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作者：
Benetka, Wolfgang;Koranda, Manfred;Maurer-Stroh, Sebastian;Pittner, Fritz;Eisenhaber, Frank
用于验证蛋白质底物通过法尼基化或香叶基香叶基化进行翻译后修饰的可用体外和体内方法（例如，用3 H-标记的锚前体进行放射自显影）耗时（数周/数月）、费力并且灵敏度低。我们描述了一种新的技术，用于检测异戊二烯锚在N-末端谷胱甘肽S-转移酶（GST）标记的目标蛋白在兔网织红细胞裂解物中体外表达的结构，并与3 H-标记的锚前体孵育。或者，可以使用在体内（在细胞培养物中）表达的血凝素（HA）标记的构建体。对于放射性标记物的注册，我们建议使用薄层色谱（TLC）分析仪。作为对照，通过在先前已用于TLC扫描的相同膜上用抗GST-（或抗HA-）抗体进行蛋白质印迹来测试蛋白质产率。这些方案已经用Rap 2A、v-Ki-Ras 2和RhoA（变体RhoA 63 L）进行了测试，包括必要的对照。我们直接表明，RasD 2是一个法尼基化目标。节省时间的实验和检测3 H标记的脂质锚的更高的灵敏度推荐的TLC扫描方法与纯化的GST-（或HA-）标记的靶蛋白作为选择的方法，用于分析其异戊烯化能力在体外和体内，也可能用于研究肉豆蔻酰和棕榈酰翻译后修饰。
Available in vitro and in vivo methods for verifying protein substrates for posttranslational modifications via farnesylation or geranylgeranylation (for example, autoradiography with 3H-labeled anchor precursors) are time consuming (weeks/months), laborious and suffer from low sensitivity. We describe a new technique for detecting prenyl anchors in N-terminally glutathione S-transferase (GST)-labeled constructs of target proteins expressed in vitro in rabbit reticulocyte lysate and incubated with 3H-labeled anchor precursors. Alternatively, hemagglutinin (HA)-labeled constructs expressed in vivo (in cell culture) can be used. For registration of the radioactive marker, we propose to use a thin layer chromatography (TLC) analyzer. As a control, the protein yield is tested by Western blotting with anti-GST- (or anti-HA-) antibodies on the same membrane that has been previously used for TLC-scanning. These protocols have been tested with Rap2A, v-Ki-Ras2 and RhoA (variant RhoA63L) including the necessary controls. We show directly that RasD2 is a farnesylation target. Savings in time for experimentation and the higher sensitivity for detecting 3H-labeled lipid anchors recommend the TLC-scanning method with purified GST- (or HA-) tagged target proteins as the method of choice for analyzing their prenylation capabilities in vitro and in vivo and, possibly, also for studying the myristoyl and palmitoyl posttranslational modifications.