Development of a minimal cell-free translation system for the synthesis of presecretory and integral membrane proteins
Development of a minimal cell-free translation system for the synthesis of presecretory and integral membrane proteins
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DOI：
10.1021/bp049553u
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发表时间：
2005-07-01
影响因子：
2.9
通讯作者：
Ueda, T
中科院分区：
文献类型：
--
作者：
Kuruma, Y;Nishiyama, K;Ueda, T
通过将翻译系统和膜整合/转位系统相结合，我们构建了一种新的无细胞系统，用于体外生产存储前期和完整的膜蛋白。在一个完全确定的、由最少数量的翻译因子组成的无细胞系统中，添加了从大肠杆菌中制备的尿素洗涤的倒置膜小泡(U-InVS)，以及介导膜靶向存储前期和完整膜蛋白的纯化蛋白。最初，仅通过添加SecA和SecB就可以获得有效的膜转位，该蛋白是一种存储前期蛋白(Pompa)。蛋白酶K消化清楚地显示了Pompa在囊泡内的成功转位。接下来，在只有SRP(FFH)和SR(FtsY)存在的情况下，内膜蛋白(MtlA)被整合到U-InVS中。最后，一个具有较大周质区域(FtsQ)的膜蛋白也被正确地整合到这个无细胞系统中，因此需要两个因子(SRP/SR和SecA/SecB)来进行膜整合/转位。因此，我们的新型无细胞系统不仅为膜相关蛋白的生产提供了一个有效的策略，而且为蛋白质转运和整合机制的生物学研究提供了一个改进的平台。
By combining translation and membrane integration/translocation systems, we have constructed a novel cell-free system for the production of presecretory and integral membrane proteins in vitro. A totally defined, cell-free system reconstituted from a minimal number of translation factors was supplemented with urea-washed inverted membrane vesicles (U-INVs) prepared from Escherichia coli, as well as with purified proteins mediating membrane targeting of presecretory and integral membrane proteins. Initially, efficient membrane translocation of a presecretory protein (pOmpA) was obtained simply by the addition of only SecA and SecB. Proteinase K digestion clearly showed the successful translocation of pOmpA inside the vesicles. Next, integration of an inner membrane protein (MtlA) into U-INVs was achieved in the presence of only SRP (Ffh) and SR (FtsY). Finally, a membrane protein possessing a large periplasmic region (FtsQ) and therefore requiring both factors (SRP/SR and SecA/SecB) for membrane integration/translocation was also shown to be integrated correctly in this cell-free system. Thus, our novel cell-free system provides not only an efficient strategy for the production of membrane-related proteins but also an improved platform for the biological study of protein translocation and integration mechanisms.