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正确折叠的能力。由于这个原因，并且由于MBP融合蛋白在E.大肠杆菌，我们已经取得了MBP的基石，我们的方法，高通量蛋白质表达和纯化。然而，MBP融合蛋白并不总是有效地与直链淀粉树脂结合，即使它们结合，融合蛋白在直链淀粉亲和层析后也很少是纯的。因此，为了补偿MBP作为亲和标签的相对较差的性能，我们尝试在MBP融合蛋白的一般框架内掺入补充标签。我们确定了MBP融合蛋白框架内的几个位置，其中可以添加辅助标签，而不损害MBP促进其融合伴侣溶解度的能力。然后，我们设计并成功地测试了一个通用的协议，蛋白质生产在E。利用双His 6-MBP亲和标签的大肠杆菌。MBP部分提高了产量并增强了乘客蛋白的溶解度，而His标签促进了其纯化。我们目前正致力于将这种方法应用于其他宿主中进行异源蛋白表达。由于大多数亲和标签都有可能干扰结构研究，因此绝对需要可靠的方法来去除它们。因此，我们已经投入了大量的努力，试图利用高度特异性的烟草蚀纹病毒（TEV）蛋白酶用于此目的。为了提高TEV蛋白酶在E.在大肠杆菌中，我们设计了一种表达载体，其以MBP融合蛋白的形式产生酶，该MBP融合蛋白在体内切割自身以产生不含MBP的N末端His标记的TEV蛋白酶催化结构域。通过使用tRNA辅助质粒来补偿大肠杆菌中很少使用的精氨酸密码子的存在，实现了TEV蛋白酶产量的显着增加。杆菌我们还设计了一种简单的方法，通过TEV蛋白酶的融合蛋白的细胞内加工，这是用来确定是否乘客蛋白是可能被正确折叠时，它融合到MBP。我们已经表明，许多不同的氨基酸侧链可以容纳在P1？在一个实施方案中，所述方法包括在TEV蛋白酶识别位点的一个或多个位点上对加工效率几乎没有影响或没有影响。因此，在许多情况下，有可能使用TEV蛋白酶来产生没有非天然残基连接到其N-末端的重组蛋白。野生型TEV蛋白酶在特定位点切割自身以产生酶活性大大降低的截短多肽。我们设法克服自溶问题，通过构建一个突变体酶（S219 V），几乎是不受自动失活和几乎两倍的催化活性的野生型酶。我们已将S219 V TEV蛋白酶表达载体分发给全球数百个研究实验室。我们还确定了TEV蛋白酶与肽底物和抑制剂复合的晶体结构，这揭示了其严格序列特异性的结构基础。我们目前正专注于其他高度特异性的蛋白酶，如烟草脉斑驳病毒（TVMV），这可能被证明是有用的替代TEV蛋白酶编码的特性。

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. 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 may prove to be useful alternatives to TEV protease.