Protein Expression and Purification in the Fast Lane

蛋白质表达和纯化的快车道

• 批准号: 8175311
• 负责人: David S Waugh
• 金额: $ 39.1万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8175311
• 关键词: Affinity; Affinity Chromatography; Alphavirus; Amino Acids; Amylose; Arginine; Autolysis; Baculovirus Expression System; Baculoviruses; Binding; C-terminal; Carboxypeptidase; Carboxypeptidase A; Catalytic Domain; Cattle; Cells; Chimeric Proteins; Cleaved cell; Codon Nucleotides; Complex; Crystallography; Cytosol; Environment; Enzymes; Escherichia coli; Future; Generic Drugs; Goals; Green Fluorescent Proteins; Kluyveromyces; Laboratory Research; Leishmania; Location; Methods; Minor; Modification; Mutation; Peptide Hydrolases; Peptides; Performance; Plant Resins; Plasmids; Procedures; Process; Production; Protein Disulfide Isomerase; Proteins; Protocols documentation; Reagent; Recombinant Proteins; Recombinants; Semliki forest virus; Side; Sindbis Virus; Site; Solubility; Specificity; Structure; Substrate Specificity; System; TEV protease; Tertiary Protein Structure; Testing; Tobacco; Transfer RNA; Veins; Venezuelan Equine Encephalitis Virus; Virus; Yeasts; base; design; enzyme substrate; expression vector; extracellular; improved; in vivo; inhibitor/antagonist; maltose-binding protein; mutant; periplasm; polyhistidine; polypeptide; protein expression; research study; structural biology; tool

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. During FY2009, we have been testing this method for the production of recombinant proteins (both secreted and intracellular formats) in the yeast Kluyveromyces lactis. We have found that the yield of intracellular HisMBP fusion proteins is extremely poor in general, and secreted HisMBP fusion proteins are frequently truncated after the MBP domain. Future experiments will explore the impact of alternative fusion partners on the yield of recombinant proteins in K. lactis. Promising results have recently been obtained (2010) using green fluorescent protein as a fusion parter for both intracellular and extracellular (secretion) protein expression in K. lactis. Experiments to develop and test tools for the production of recombinant proteins in the protozoan Leishmania tarantolae are just getting underway, as is an effort to evaluate the host Brevibacillus choshinensis for the secretion of recombinant proteins. 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 characterized a trio of alphavirus proteases encoded by Sindbis Virus, Semliki Forest Virus and Venezuelan Equine Encephalitis Virus that we hoped would be useful alternatives to TEV protease. Although these proteases were shown to have adequate specificity for use as reagents to remove affinity tags, their catalytic efficiency is far less than that of the potyviral TEV and TVMV proteases. Finally, we have been investigating the utility of a recombinant form of a fungal carboxypeptidase (MeCPA) for removing short affinity tags (e.g., polyhistidine) from the C-termini of recombinant proteins. We have carried out a thorough analysis of the enzyme's substrate specificity and shown that it is capable of being used to remove C-terminal His-tags on a preparative level for crystallography. However, the yield of MeCPA obtained from the baculovirus expression system is rather low (250 micrograms per liter), and so efforts are underway to find a more efficient way to produce the recombinant enzyme. Alternative enzymes, such as bovine carboxypeptidases A and B are also being expressed in a variety of systems. We recently (2010) developed a method for the production of MeCPA in E. coli that produces twice the yield that can be obtained from the baculovirus system, and it seems likely that substantially greater yields will can be achieved with minor modifications of the current procedure. The key was to express the enzyme as an MBP fusion protein in E. coli cells that carry mutations that transform the cytosol into a more oxidative environment, while simultaneously overproducing the protein disulfide isomerase DsbC, which normally resides in the periplasm, in the cytosol.

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。在2009财年期间，我们一直在测试这种方法，用于生产酵母kluyveromyces乳酸中的重组蛋白（分泌和细胞内格式）。我们发现，细胞内HISMBP融合蛋白的产率一般都非常差，并且分泌的HISMBP融合蛋白经常在MBP结构域之后被截断。未来的实验将探索替代融合伙伴对乳酸乳乳糖蛋白重组蛋白产量的影响。最近（2010年）使用绿色荧光蛋白作为乳酸乳杆菌中细胞内和细胞外（分泌）蛋白表达的融合蛋白作为融合蛋白获得了有希望的结果。开发和测试用于生产原生动物利什曼原虫塔兰氏菌中重组蛋白的实验正在进行中，这是为了评估宿主brevibacillus choshinensis的重组蛋白分泌的努力。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）来克服自溶的问题，该酶几乎没有自动激活，几乎是催化活性的两倍，就像野生型酶一样活跃。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，我们表征了由Sindbis病毒，Semliki Forest病毒和委内瑞拉马匹脑炎病毒编码的三重奏α病毒蛋白酶，我们希望我们希望这些病毒是TEV蛋白酶的有用替代方法。尽管这些蛋白酶被证明具有足够的特异性，可作为试剂去除亲和力标签，但它们的催化效率远远低于波基病毒TEV和TVMV蛋白酶的催化效率。最后，我们一直在研究一种真菌羧肽酶（MECPA）重组形式的实用性，用于从重组蛋白的C末端去除短亲和标签（例如多组氨酸）。我们已经对酶的底物特异性进行了彻底的分析，并表明它能够在制备水平上去除C末端HIS标签以进行晶体学。但是，从杆状病毒表达系统获得的MECPA产量相当低（每升250微克），因此正在努力寻找一种更有效的方法来产生重组酶。替代酶，例如牛羧肽酶A和B，也在各种系统中表达。我们最近（2010年）开发了一种在大肠杆菌中生产MECPA的方法，该方法产生的两倍是可以从杆状病毒系统中获得的产量，并且对于当前程序的较小修改，似乎可以实现更大的产量。 。关键是将酶表示为大肠杆菌细胞中的MBP融合蛋白，该突变携带突变，可将细胞质转化为更氧化的环境，同时过量产生蛋白质二硫化物异构酶DSBC，通常驻留在细胞质中。