Protein Expression and Purification in the Fast Lane

该项目最近在两个领域取得了值得注意的进展：了解麦芽糖结合蛋白（MBP）增强溶解性和促进其融合伴侣折叠的机制，以及开发羧肽酶作为去除亲和标签的试剂。大肠杆菌麦芽糖结合蛋白（MBP）在促进其融合伴侣的溶解性方面是不常见的有效的。为了研究MBP溶解度增强的机制，我们比较了MBP融合蛋白在体外复性与在天然条件下纯化的相应融合蛋白的性质。将五种乘客蛋白融合到3种不同的N-末端标签：His 6-MBP、His 6-GST和His 6。15个融合蛋白在变性条件下纯化，然后通过快速稀释复性。在所有的情况下，我们回收了更多的可溶性MBP融合蛋白比可溶性GST或His标记的蛋白复性后。因此，我们可以在一个简单的体外系统中重现MBP的增溶活性，表明不需要额外的因素来介导这种效果。我们测定了可溶性融合蛋白及其TEV蛋白酶消化产物（即去除N-末端标签）的生物活性。对于一些融合蛋白，几乎没有或没有检测到活性，而其他融合蛋白则相当有活性。从E.在天然条件下的大肠杆菌中，检测到所有乘客蛋白的实质活性。这些结果表明，MBP的能力，以促进其融合配偶体在体外的溶解性，有时，但并不总是，在其正确的折叠结果。我们发现，一些乘客蛋白的折叠是由内源性伴侣在体内介导的。因此，MBP在折叠过程中充当被动参与者;乘客蛋白自发折叠或在分子伴侣的帮助下折叠。因此，工程改造的MBP融合蛋白可以模拟存在于许多真核蛋白中的天然顺式作用分子伴侣结构域的作用。我们一直在研究重组形式的真菌羧肽酶（MeCPA）用于去除短亲和标签（例如，多聚组氨酸）。我们已经进行了彻底的分析酶的底物特异性，并表明它是能够被用来删除C-末端组氨酸标签上的晶体学制备水平。然而，从杆状病毒表达系统获得的MeCPA的产率相当低（250微克/升），因此正在努力寻找更有效的方法来生产重组酶。替代酶，如牛羧肽酶A和B也在多种系统中表达。我们最近（2011年）开发了一种在大肠杆菌中生产MeCPA的方法。大肠杆菌的产量是从杆状病毒系统获得的产量的两倍，并且似乎有可能通过对当前方法的微小修改来实现实质上更高的产量。关键是在大肠杆菌中表达MBP融合蛋白。携带突变的大肠杆菌细胞，这些突变将细胞质转化为更氧化的环境，同时在细胞质中过量产生蛋白质二硫键异构酶DsbC，该酶通常存在于周质中。同样的方法最近已被用于在大肠杆菌中产生活性牛羧肽酶B。杆菌目前正在进行实验，以测试A型和B型羧肽酶对球状蛋白底物的“鸡尾酒”，以建立该方法的原理证明，并进一步了解酶的特异性。最近，两个细菌羧肽酶从Thermoactinomycesvulgarism和Streptomycesgresius，已报道表现出双重A-和B-型特异性已被克隆，其生化特性正在调查中。

Noteworthy progress on this project has been made recently in two areas: understanding the mechanism by which maltose-binding protein (MBP) enhances the solubility and promotes the folding of its fusion partners, and the development of carboxypeptidases as reagents for the removal of affinity tags. Escherichia coli maltose binding protein (MBP) is uncommonly effective at promoting the solubility of its fusion partners. To investigate the mechanism of solubility enhancement by MBP, we compared the properties of MBP fusion proteins refolded in vitro with those of the corresponding fusion proteins purified under native conditions. Five passenger proteins were fused to 3 different N-terminal tags: His6-MBP, His6-GST and His6. The 15 fusion proteins were purified under denaturing conditions and then refolded by rapid dilution. In all cases we recovered far more soluble MBP fusion protein than soluble GST- or His-tagged protein after refolding. Hence, we can reproduce the solubilizing activity of MBP in a simple in vitro system, indicating that no additional factors are required to mediate this effect. We assayed both the soluble fusion proteins and their TEV protease digestion products (i.e. with the N-terminal tag removed) for biological activity. Little or no activity was detected for some fusion proteins whereas others were quite active. When the MBP fusion proteins were purified from E. coli under native conditions, substantial activity was detected for all of the passenger proteins. These results indicate that the ability of MBP to promote the solubility of its fusion partners in vitro sometimes, but not always, results in their proper folding. We show that the folding of some passenger proteins is mediated by endogenous chaperones in vivo. Hence, MBP serves as a passive participant in the folding process; passenger proteins either fold spontaneously or with the assistance of chaperones. Engineered MBP fusion proteins may therefore mimic the action of natural cis-acting chaperone domains that are present in many eukaryotic proteins. 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 (2011) 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. The same approach has recently been used to produce active bovine carboxypeptidase B in E. coli. Experiments are currently underway to test a "cocktail" of A- and B-type carboxypeptidases on globular protein substrates to establish proof of principle for the method and gain further insight into the specificity of the enzymes. Most recently, two bacterial carboxypeptidases from Thermoactinomyces vulgarism and Streptomyces gresius that have been reported to exhibit dual A- and B-type specificities have been cloned and their biochemical properties are under investigation.