Difference between revisions of "Part:BBa K3733038"

Revision as of 17:16, 19 October 2021

ScGS with a His-tag

Gesomin synthase from Streptomyces coelicolor A3(2) (ScGS) is a single 726-amino acid protein which catalyzes the Mg²⁺ dependent conversion of farnesyl diphosphate to a mixture including geosmin. A 6×His tag is added in its C-terminal to make it accessible for Ni-NTA purification.

Usage and Biology

The ScGS is a bifunctional sesquiterpene cyclase, with the presence of Mg²⁺, the N-terminal half of this protein catalyzes the ionization and cyclization of farnesyl diphosphate to form germacradienol and inorganic pyrophosphate(PP_i). Then the C-terminal domain, highly homologous with the former, catalyzes the protonation, cyclization, and fragmentation of germacradienol to form geosmin and acetone.

Sequence and Features

Functional Parameters

To obtain the protein, pET-28a(+)-ScGS(with His-tag) was transferred into E.coli BL21(DE3), and the cells were inoculated in 25 mL cultures of LB medium with 10 μg/mL kanamycin. These cultures were grown at 37℃ with 250 rpm shaking until the OD₆₀₀ reached 0.5-0.8, then 0.3 mM isopropyl β-D-1-thiogalactopyranoside(IPTG) were added, following by an overnight cultivation at 16℃ with 250 rpm shaking to induce protein expression. The washed and harvested cells were resuspended with a Binding Buffer, and then the cells were lysed by ultrasonication. Purification was performed according to the protocol of Ni-NTA Sefinose^TM Resin (Sangon Biotech, Shanghai, China). As it shows in the following figure(figure 1.), the existence of with a 6×His tag in our chasis was clearly proved by SDS-PAGE analysis.

References

[1] Harris G G, Lombardi P M, Pemberton T A, et al. Structural Studies of Geosmin Synthase, a Bifunctional Sesquiterpene Synthase with αα Domain Architecture That Catalyzes a Unique Cyclization–Fragmentation Reaction Sequence[J]. Biochemistry, 2015, 54(48): 7142-7155.