Protein Expression and Purification in the Fast Lane

蛋白质表达和纯化的快车道

基本信息

批准号：
7733010
负责人：
David S Waugh
金额：
$ 42.72万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

项目摘要

We previously demonstrated that E. coli maltose binding protein (MBP) has a remarkable ability to enhance the solubility and promote the proper folding of its fusion partners. For this reason, and because MBP fusion proteins routinely accumulate to very high levels in E. coli, we have made MBP the cornerstone of our approach for high-throughput protein expression and purification. However, MBP fusion proteins do not always bind efficiently to amylose resin, and even when they do the fusion proteins are rarely pure after amylose affinity chromatography. Therefore, to compensate for the relatively poor performance of MBP as an affinity tag, we attempted to incorporate supplementary tags within the general framework of an MBP fusion protein. We identified several locations within the framework of an MBP fusion protein where accessory tags could be added without compromising the ability of MBP to promote the solubility of its fusion partners. We then designed and successfully tested a generic protocol for protein production in E. coli that utilizes a dual His6-MBP affinity tag. The MBP moiety improves the yield and enhances the solubility of the passenger protein while the His-tag facilitates its purification. We are currently working on applying this method in other hosts for heterologous protein expression, beginning with the yeast K. lactis. Because most affinity tags have the potential to interfere with structural studies, reliable ways to remove them are absolutely necessary. Accordingly, we have invested a substantial effort in trying to exploit the highly specific tobacco etch virus (TEV) protease for this purpose. To improve the solubility of TEV protease in E. coli, we designed an expression vector that produces the enzyme in the form of an MBP fusion protein that cleaves itself in vivo to generate an N-terminally His-tagged TEV protease catalytic domain that is free of MBP. A dramatic increase in the yield of TEV protease was realized by using a tRNA accessory plasmid to compensate for the presence of arginine codons that are rarely used in E. coli. We also devised a simple method for intracellular processing of fusion proteins by TEV protease, which is used to determine whether or not a passenger protein is likely to be properly folded when it is fused to MBP. We have shown that many different amino acid side chains can be accommodated in the P1' site of a TEV protease recognition site with little or no impact on the efficiency of processing. Consequently, in many cases it is possible to use TEV protease to produce recombinant proteins with no non-native residues attached to their N-termini. Wild-type TEV protease cleaves itself at a specific site to generate a truncated polypeptide with greatly reduced enzymatic activity. We managed to overcome the autolysis problem by constructing a mutant enzyme (S219V) that is nearly impervious to autoinactivation and almost twice as catalytically active as the wild-type enzyme. We have distributed S219V TEV protease expression vectors to hundreds of research laboratories around the world. We have also determined crystal structures of TEV protease complexed with a peptide substrate and an inhibitor, which revealed the structural basis of its stringent sequence specificity. We are currently focusing on the characterization of other highly specific proteases, such as that encoded by the tobacco vein mottling virus (TVMV), which we have recently crystallized in complex with a peptide substrate. The co-crystal structure suggested that TVMV protease should have more stringent sequence specificity in the S1' pocket, and we have been able to confirm this experimentally. More recently, we have begun to characterize alphavirus proteases, such as those encoded by Sindbis Virus, Semliki Forest Virus and Venezuelan Equine Encephalitis Virus, which may prove to be useful alternatives to TEV protease. Finally, we are investigating the utility of a recombinant form of a fungal carboxypeptidase for removing short affinity tags (e.g., polyhistidine) from the C-termini of proteins.

我们先前证明了大肠杆菌麦芽糖结合蛋白（MBP）具有提高溶解度并促进其融合伴侣的正确折叠的出色能力。因此，由于MBP融合蛋白在大肠杆菌中常规积累至非常高的水平，因此我们使MBP成为了高通量蛋白表达和纯化方法的基石。但是，MBP融合蛋白并不总是有效地与双淀粉树脂结合，即使它们进行融合蛋白在链淀粉亲和色谱后也很少纯净。因此，为了补偿MBP作为亲和力标签的相对较差的性能，我们试图将补充标签纳入MBP融合蛋白的一般框架中。我们在MBP融合蛋白的框架内确定了几个位置，可以在其中添加附件标签，而不会损害MBP促进其融合伙伴的溶解度的能力。然后，我们设计并成功地测试了使用双重HIS6-MBP亲和力标签的大肠杆菌中蛋白质生产的通用方案。 MBP部分可提高产量并增强乘客蛋白的溶解度，而HIS标签则促进其纯化。我们目前正在努力在其他宿主中应用此方法以进行异源蛋白表达，从酵母K.乳酸乳酸乳酸开始。由于大多数亲和力标签有可能干扰结构研究，因此绝对必要的可靠方法去除它们。因此，为此，我们投入了大量努力来试图利用高度特定的烟草蚀刻病毒（TEV）蛋白酶。为了提高TeV蛋白酶在大肠杆菌中的溶解度，我们设计了一种表达载体，该表达载体以MBP融合蛋白的形式产生酶，该酶在体内裂解以产生N末端His His标记的TEV蛋白酶催化结构域，这是Free MBP。通过使用tRNA辅助质粒来补偿很少在大肠杆菌中使用的精氨酸密码子的存在来实现TEV蛋白酶产量的显着提高。我们还设计了一种简单的方法，用于通过TEV蛋白酶对融合蛋白进行细胞内加工，该方法用于确定当乘客蛋白与MBP融合时是否可以正确折叠乘客蛋白。我们已经表明，可以在TEV蛋白酶识别位点的P1'部位容纳许多不同的氨基酸侧链，对加工效率的影响很小或没有影响。因此，在许多情况下，可以使用TEV蛋白酶产生重组蛋白，而没有附着在其N末端的非本性残基。野生型TEV蛋白酶在特定部位裂解，产生截短的多肽，其酶活性大大降低。我们通过构建一种突变酶（S219V）来克服了自溶的问题，该酶几乎不受自动活化的影响，几乎是催化活性的两倍，是野生型酶的催化活性。我们已经将S219V TEV蛋白酶表达向量分发给了世界上数百个研究实验室。我们还确定了与肽底物和抑制剂复合的TEV蛋白酶的晶体结构，这揭示了其严格序列特异性的结构基础。我们目前正在关注其他高度特异性蛋白酶的表征，例如由烟草静脉斑驳病毒（TVMV）编码的蛋白酶，我们最近与肽底物结晶了。共晶结构表明，TVMV蛋白酶在S1'袋中应该具有更严格的序列特异性，我们已经能够通过实验确认此。最近，我们已经开始表征α病毒蛋白酶，例如Sindbis病毒，Semliki Forest病毒和委内瑞拉马脑炎病毒所编码的α蛋白酶，这可能被证明是TEV蛋白酶的有用替代方法。最后，我们正在研究一种真菌羧肽酶重组形式的实用性，以从蛋白质的C-末端去除短亲和标签（例如多组氨酸）。