Intein-mediated synthesis of geranylgeranylated Rab7 protein in vitro
Intein-mediated synthesis of geranylgeranylated Rab7 protein in vitro
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DOI：
10.1021/ja017799e
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发表时间：
2002-05-22
影响因子：
15
通讯作者：
Waldmann, H
中科院分区：
文献类型：
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作者：
Alexandrov, K;Heinemann, I;Waldmann, H
一般来说，重组蛋白的生产是生物技术和生命科学的重要先决条件。然而，用于生产翻译后修饰的重组蛋白或具有非天然官能团（例如荧光团、自旋标记等）的蛋白的方法很少。在这项工作中，我们结合使用有机合成和体外蛋白质连接来构建单异戊二烯化 Rab7 GTPase。该蛋白质由重组 N 端部分和模拟 Rab7 C 端的肽制备。为了构建合成的六个氨基酸长的荧光单异戊二烯化肽，我们使用了嵌段缩合策略。连接的产率>70%。通过有机萃取、相分配和重折叠的组合，从未连接的肽中纯化所得蛋白质。重折叠的单异戊二烯化半合成 Rab7 蛋白 (Rab7GG) 与其天然伴侣 REP-1（Rab 护送蛋白 1）形成稳定的复合物，并且可以作为酶促异戊二烯化反应中第二个异戊二烯基的受体。使用荧光光谱，我们表征了 Rab7GG:REP-1 复合物与 Rab 香叶基香叶基转移酶的相互作用，并得出结论，它是异戊二烯化反应的真正中间体。因此，我们提出了使用天然化学连接方法体外生成半合成脂化蛋白的第一个例子。
Production of recombinant proteins is an important prerequisite for biotechnology and life sciences in general. However, there is a paucity of methods for production of posttranslationally modified recombinant proteins or proteins with non-native functional groups, such as fluorophores, spin labels, and so forth. In this work we have used a combination of organic synthesis and in vitro protein ligation to construct monoprenylated Rab7 GTPase. The protein was prepared from a recombinant N-terminal portion and a peptide mimicking the C terminus of Rab7. For construction of a synthetic six-amino-acid-long fluorescent monoprenylated peptide, we used a block condensation strategy. Ligation was achieved with a yield of >70%. The resulting protein was purified from the unligated peptide by a combination of organic extraction and phase partitioning and refolding. The refolded monoprenylated semisynthetic Rab7 protein (Rab7GG) formed a stable complex with its natural chaperone REP-1 (Rab escort protein 1) and could serve as an acceptor of the second prenyl group in the enzymatic prenylation reaction. Using fluorescence spectroscopy, we characterized the interaction of the Rab7GG:REP-1 complex with Rab geranylgeranyl transferase and came to the conclusion that it functioned as a genuine intermediate of the prenylation reaction. Thus, we present the first example of the in vitro generation of a semisynthetic lipidated protein using the native chemical ligation method.