Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency

Abstract

Because of its stringent sequence specificity, the catalytic domain of the nuclear inclusion protease from tobacco etch virus (TEV) is a useful reagent for cleaving genetically engineered fusion proteins. However, a serious drawback of TEV protease is that it readily cleaves itself at a specific site to generate a truncated enzyme with greatly diminished activity. The rate of autoinactivation is proportional to the concentration of TEV protease, implying a bimolecular reaction mechanism. Yet, a catalytically active protease was unable to convert a catalytically inactive protease into the truncated form. Adding increasing concentrations of the catalytically inactive protease to a fixed amount of the wild-type enzyme accelerated its rate of autoinactivation. Taken together, these results suggest that autoinactivation of TEV protease may be an intramolecular reaction that is facilitated by an allosteric interaction between protease molecules. In an effort to create a more stable protease, we made amino acid substitutions in the P2 and P1' positions of the internal cleavage site and assessed their impact on the enzyme's stability and catalytic activity. One of the P1' mutants, S219V, was not only far more stable than the wild-type protease (approximately 100-fold), but also a more efficient catalyst.

Making the most of affinity tags

“…This has always been the Achilles' heel of the fusion approach. Although highly specific endoproteases, such as those encoded by the tobacco etch virus (AcTEV, Invitrogen) [29] and the human rhinovirus (PreScission, Amersham Biotech.) [30], have largely mitigated the problem of nonspecific cleavage, processing efficiency varies with each fusion protein in an unpredictable manner.…”

“…The MBP moiety improves the yield and enhances the solubility of the passenger protein while the His 6 -tag facilitates its purification. The fusion protein (His 6 -MBP-passenger) is purified by IMAC on Ni-NTA resin and then cleaved in vitro with His 6 -tagged TEV protease [29] to separate the His 6 -MBP from the passenger protein. In the final step, the unwanted byproducts of the digest (His 6 -MBP, His 6 -TEV protease, and any undigested fusion protein)…”

Atomic resolution structure of the cytoplasmic domain of Yersinia pestis YscU, a regulatory switch involved in type III secretion

“…The lysate was centrifuged at 15,000 rpm at 4 C using an SA-600 rotor, filtered, and then the HisMBP fusion proteins were purified by immobilized metal affinity chromatography (IMAC) as described. 20 Fractions containing the fusion proteins were pooled, cleaved overnight with hexahistidine-tagged TEV protease, 22 and then subjected to another round of IMAC as described. 20 The flow-through fractions containing YscU were pooled and applied to an amylose column (New England Biolabs, Beverely, MA) to remove a small amount of His 6 -MBP that did not bind to the second Ni-NTA column.…”

An expression vector tailored for large-scale, high-throughput purification of recombinant proteins

“…Insertion of the gene encoding MBP, amplified by PCR from the vector pRK739 [17], into the leader sequence encoding region of pMCSG7 gave pMCSG9 (Fig. 1, Materials and methods).…”

“…The MBP encoding region was generated by PCR using plasmid pRK793 [17] as template (a generous gift from David Waugh) and the primers 5′-TTTTAGATCTGATGTCCCCTATACTAGGTTATTGG and 5′-TTTTGGTACCTGGGATATCGTAATCATCCGATTTTGGAGGATGGT (purchased from the Howard Hughes Medical Institute-Keck Laboratory of Yale University, New Haven, CT). The vector was digested with KpnI and dephosphorylated with calf intestinal phosphatase (Promega, Madison, WI), and ligated to the KpnI-treated PCR product.…”