Protein Expression and Purification in the Fast Lane

我们先前已经证明，大肠杆菌麦芽糖结合蛋白(MBP)具有显著的提高其融合蛋白的溶解性和促进其正确折叠的能力。出于这个原因，由于MBP融合蛋白在大肠杆菌中通常积累到非常高的水平，我们将MBP作为我们高通量蛋白表达和纯化的基石。然而，MBP融合蛋白并不总是有效地结合到直链淀粉树脂上，即使它们有效地结合在一起，经过直链淀粉亲和层析后的融合蛋白也很少是纯净的。因此，为了弥补MBP作为亲和标签的相对较差的性能，我们试图将补充标签整合到MBP融合蛋白的一般框架中。我们确定了MBP融合蛋白框架内的几个位置，在这些位置可以添加辅助标签，而不会影响MBP促进其融合伙伴溶解的能力。然后，我们设计并成功地测试了一种在大肠杆菌中生产蛋白质的通用方案，该方案利用了双重His6-MBP亲和标签。MBP部分提高了乘客蛋白的得率和溶解性，而His-Tag有助于其纯化。我们目前正致力于将这种方法应用于其他宿主的异源蛋白表达。由于大多数亲和标签都有可能干扰结构研究，因此可靠的方法去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，它以MBP融合蛋白的形式产生这种酶，该融合蛋白在体内自我切割，从而产生一个N末端标记的TEV蛋白酶催化结构域，该催化结构域不含MBP。TEV蛋白酶产量的显著提高是通过使用tRNA辅助质粒来补偿在大肠杆菌中很少使用的精氨酸密码子的存在实现的。我们还设计了一种利用TEV蛋白酶在细胞内处理融合蛋白的简单方法，该方法用于确定乘客蛋白在与MBP融合时是否可能正确折叠。我们已经证明，许多不同的氨基酸侧链可以容纳在P1？TEV蛋白酶识别位点的位置，对处理效率影响很小或没有影响。因此，在许多情况下，可以使用TEV蛋白酶来产生N-末端没有非天然残基的重组蛋白。野生型TEV蛋白酶在特定的位置裂解自己，产生一种截短的多肽，其酶活性大大降低。我们成功地克服了自溶问题，构建了一种几乎不受自失活影响的突变酶(S219V)，其催化活性几乎是野生型酶的两倍。我们已经将S219V TEV蛋白酶表达载体分发到世界各地的数百个研究实验室。我们还测定了TEV蛋白酶与多肽底物和抑制剂的络合物的晶体结构，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的蛋白酶的特性，如烟草静脉斑驳病毒(TVMV)编码的那些，这可能被证明是有用的TEV蛋白酶的替代品。

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.