Preformed GroES Oligomers Are Not Required as Functional Cochaperonins

We have previously shown that the C-terminal sequence of GroES is required for oligomerization [Seale and Horowitz (1995), J. Biol. Chem. 270, 30268–30270]. In this report, we have generated a C-terminal deletion mutant of GroES with a significantly destabilized oligomer and have investigated its function in the chaperonin-assisted protein folding cycle. Removal of the two C-terminal residues of GroES results in a cochaperonin [GroESD(96–97)] that is monomeric at concentrations where GroES function is assessed. Using equilibrium ultracentrifugation, we measured the dissociation constant for the oligomer–monomer equilibrium to be 7.3×10^–34M⁶. The GroESD(96–97) is fully active as a cochaperonin. This mutant is able to inhibit the ATPase activity of GroEL to levels comparable to wild-type GroES. It is also able to assist the refolding of urea-denatured rhodanese by GroEL. While GroESD(96–97) can function at levels comparable to wild-type GroES, higher concentrations of mutant are required to produce the same effect. These results support the idea that the preformed GroES heptamer is not required for function, but they suggest that the oligomeric cochaperonin is most efficient.     

Improved Large-Scale Preparation of Phage T4 Endonuclease VII Overexpressed in E. coli

Using PCR, we cloned T4 gene 49, which encodes the endonucleaseVII, and the inactive mutant gene 49 amE727 into vector pET-11a.In combination with Escherichia coli host strain BL21(DE3),this system provided excellent repression of the expressionof the highly toxic protein before induction with IPTG. Afterinduction, the proteins were made in high quantities while remainingsoluble. Dilution of the crude lysate at 1 : 10,000 continuedto show a highly specific activity in the case of the wild-typeenzyme. The protein was purified to homogeneity with a recoveryof 33% using two chromatography steps. The yield was 20 timeshigher and the specific activity 500 times higher than thatobtained by using the previously published protocol.