Protein Expression and Purification in the Fast Lane

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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.