Protein Expression and Purification in the Fast Lane

我们以前证明了E.大肠杆菌麦芽糖结合蛋白（MBP）具有显著的 增强溶解性和促进其融合配偶体的正确折叠的能力。为 这是因为MBP融合蛋白在E. 大肠杆菌中，我们已经使MBP成为我们高通量蛋白表达方法的基石 和纯化。然而，MBP融合蛋白并不总是有效地与直链淀粉结合 树脂，即使他们这样做，融合蛋白很少是纯的后，直链淀粉亲和力 层析因此，为了补偿MBP作为一种 亲和标记，我们试图将补充标记的一般框架内， MBP融合蛋白。我们在MBP融合的框架内确定了几个位置 蛋白质，其中可以添加辅助标签而不损害MBP促进 其融合伴侣的溶解度。然后，我们设计并成功测试了一种通用的 在E.利用双His 6-MBP亲和标签的大肠杆菌。的MBP 部分提高了产量并增强了乘客蛋白的溶解性，而 His标签有助于其纯化。目前，我们正在研究将这种方法应用于 其它宿主用于异源蛋白表达，从酵母K.乳酸。因为 大多数亲和标记有可能干扰结构研究， 删除它们是绝对必要的。因此，我们投入了大量的努力， 试图为此目的开发高度特异性的烟草蚀纹病毒（TEV）蛋白酶。到 提高TEV蛋白酶在E.我们设计了一个表达载体， 以MBP融合蛋白的形式产生酶，该酶在体内自我切割， 产生不含MBP的N-末端His-标记的TEV蛋白酶催化结构域。一 通过使用tRNA辅助质粒实现了TEV蛋白酶产量的显著增加 以补偿在E.杆菌我们也 设计了一种用TEV蛋白酶在细胞内加工融合蛋白的简单方法， 用于确定乘客蛋白质是否可能正确折叠， 它与MBP融合。我们已经表明，许多不同的氨基酸侧链可以被 在TEV蛋白酶识别位点的P1'位点中容纳的蛋白酶，对TEV蛋白酶识别位点的P1'位点几乎没有或没有影响。 处理的效率。因此，在许多情况下，可以使用TEV蛋白酶 以产生N-末端没有连接非天然残基的重组蛋白。 野生型TEV蛋白酶在特定位点切割自身以产生截短的多肽 酶活性大大降低。我们设法克服了自溶问题， 构建几乎不受自身失活影响且几乎 其催化活性是野生型酶的两倍。我们已经分发了S219 V TEV蛋白酶 表达载体，以数百个研究实验室在世界各地。我们还 测定了与肽底物复合的TEV蛋白酶的晶体结构， 抑制剂，揭示了其严格序列特异性的结构基础。我们 目前集中于其他高度特异性蛋白酶的表征，例如， 由烟草脉斑驳病毒（TVMV）编码，我们最近在 与肽底物复合。共晶结构表明TVMV蛋白酶应 在S1'口袋中具有更严格的序列特异性，我们已经能够证实 这个实验。最近，我们已经开始表征甲病毒蛋白酶，如 与辛德毕斯病毒、塞姆利基森林病毒和委内瑞拉马脑炎病毒编码的病毒一样， 病毒，这可能被证明是有用的替代TEV蛋白酶。最后，我们 研究重组形式的真菌羧肽酶用于去除 短亲和标签（例如，多聚组氨酸）。

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.