Protein Expression and Purification in the Fast Lane

我们之前证明了大肠杆菌麦芽糖结合蛋白（MBP）具有显著的增强溶解度和促进融合伙伴正确折叠的能力。由于这个原因，并且因为MBP融合蛋白在大肠杆菌中通常积累到非常高的水平，我们已经将MBP作为我们高通量蛋白表达和纯化方法的基石。然而，MBP融合蛋白并不总是有效地与直链淀粉树脂结合，即使他们这样做，融合蛋白在直链淀粉亲和层析后也很少是纯的。因此，为了弥补MBP作为亲和力标签相对较差的性能，我们试图在MBP融合蛋白的总体框架内加入补充标签。我们在MBP融合蛋白的框架内确定了几个位置，在这些位置上可以添加辅助标签，而不会影响MBP促进其融合伙伴溶解度的能力。然后，我们设计并成功测试了一种利用双His6-MBP亲和标签在大肠杆菌中生产蛋白质的通用方案。MBP片段提高了产率并增强了乘客蛋白的溶解度，而his标签则有助于其纯化。我们目前正致力于将这种方法应用于其他宿主，从酵母K. lactis开始进行异种蛋白的表达。因为大多数亲和标签都有可能干扰结构研究，所以去除它们的可靠方法是绝对必要的。因此，我们投入了大量的努力，试图利用高特异性烟草蚀刻病毒（TEV）蛋白酶来实现这一目的。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，以MBP融合蛋白的形式产生酶，该蛋白在体内自我裂解，产生一个n端his标记的TEV蛋白酶催化结构域，该结构域不含MBP。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，实现了TEV蛋白酶产量的显着增加。我们还设计了一种简单的方法，通过TEV蛋白酶在细胞内处理融合蛋白，用于确定当融合到MBP时，乘客蛋白是否可能被正确折叠。我们已经证明，许多不同的氨基酸侧链可以被容纳在TEV蛋白酶识别位点的P1'位点上，而对加工效率几乎没有影响。因此，在许多情况下，可以使用TEV蛋白酶来生产重组蛋白，其n端没有附着非天然残基。野生型TEV蛋白酶在一个特定的位点上自我切割，产生一个截断的多肽，酶活性大大降低。通过构建一种突变酶（S219V），我们成功地克服了自溶问题，该酶几乎不受自失活的影响，催化活性几乎是野生型酶的两倍。我们已经向全球数百个研究实验室分发了S219V TEV蛋白酶表达载体。我们还确定了TEV蛋白酶与肽底物和抑制剂络合的晶体结构，揭示了其严格序列特异性的结构基础。我们目前正专注于表征其他高度特异性的蛋白酶，例如由烟草静脉斑驳病毒（TVMV）编码的蛋白酶，我们最近将其与肽底物结合结晶。该共晶结构提示TVMV蛋白酶在S1'口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证实这一点。最近，我们已经开始表征甲病毒蛋白酶，例如由Sindbis病毒、Semliki森林病毒和委内瑞拉马脑炎病毒编码的那些，它们可能被证明是TEV蛋白酶的有用替代品。最后，我们正在研究重组形式的真菌羧肽酶的效用，用于从蛋白质的c端去除短亲和力标签（例如，多组氨酸）。

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.