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首页> 外文期刊>Protein Expression and Purification >A new Gateway (R) vector and expression protocol for fast and efficient recombinant protein expression in Mycobacterium smegmatis
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A new Gateway (R) vector and expression protocol for fast and efficient recombinant protein expression in Mycobacterium smegmatis

机译：一种新的Gateway（R）载体和表达方案，可在耻垢分枝杆菌中快速高效地表达重组蛋白

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摘要

A major obstacle associated with recombinant protein over-expression in Escherichia coli is the production of insoluble inclusion bodies, a problem particularly pronounced with Mycobacterium tuberculosis proteins. One strategy to overcome the formation of inclusion bodies is to use an expression host that is more closely related to the organism from which the proteins are derived. Here we describe methods for efficiently identifying M. tuberculosis proteins that express in soluble form in Mycobacterium smegmatis. We have adapted the M. smegmatis expression vector pYUB1049 to the Gateway(D cloning system by the addition of att recombination recognition sequences. The resulting vector, designated pDESTsing, is compatible with our in-house Gateway (R) methods for E. coli expression. A target can be subcloned into pDESTsmg by a simple LR reaction using an entry clone generated for E. coli expression, removing the need to design new primers and re-clone target DNA. Proteins are expressed by culturing the M. smegmatis strain mc(2)4517 in autoinduction media supplemented with Tween 80. The media used are the same as those used for expression of proteins in E coli, simplifying and reducing the cost of the switch to an alternative host. The methods have been applied to a set of M. tuberculosis proteins that form inclusion bodies when expressed in E. coli. We found that five of eight of these previously insoluble proteins become soluble when expressed in M. smegmatis, demonstrating that this is an efficient salvage strategy. (c) 2007 Elsevier Inc. All rights reserved.
机译：与重组蛋白在大肠杆菌中过量表达有关的主要障碍是不溶性包涵体的产生，这是结核分枝杆菌蛋白特别明显的问题。克服包涵体形成的一种策略是使用表达宿主，该宿主与衍生蛋白质的生物更为紧密相关。在这里，我们描述了有效鉴定在耻垢分枝杆菌中可溶形式表达的结核分枝杆菌蛋白的方法。我们已经通过添加att重组识别序列使耻垢分枝杆菌表达载体pYUB1049适应了Gateway（D）克隆系统。产生的载体pDESTsing与我们用于表达大肠杆菌的内部Gateway（R）方法兼容可以使用产生用于大肠杆菌表达的进入克隆，通过简单的LR反应，通过简单的LR反应将靶标亚克隆到pDESTsmg中，而无需设计新的引物并重新克隆靶标DNA。通过培养耻垢分枝杆菌菌株mc（ 2）4517在添加了Tween 80的自动诱导培养基中使用。该培养基与在大肠杆菌中表达蛋白质所使用的培养基相同，从而简化并降低了切换至其他宿主的成本。结核分枝杆菌蛋白在大肠杆菌中表达时会形成包涵体，我们发现这些先前不溶的蛋白质中有八分之五在耻垢分枝杆菌中表达时可溶，这证明这是一种有效的方法ent打捞策略。 （c）2007 Elsevier Inc.保留所有权利。

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