Independently inducible system of gene expression for condensed single protein production (cSPP) suitable for high efficiency isotope enrichment
The ability to produce isotope-enriched proteins is fundamental to the success of modern protein NMR, and is particularly essential for NMR activities in structural genomics projects. Conventional methods of protein production often prove to be cost prohibitive for obtaining samples, particularly perdeuterated and site-specifically labeled proteins. The condensed single protein production system (cSPP), providing protein expression following condensation of cells 10–40 fold, allows for the production of such samples at a fraction of the cost. The previously described cSPP system is a two plasmid system where both the MazF toxin and ACA-less target gene are coinduced with IPTG. Coinduction results in 10–20% of the target protein produced without isotopic enrichment. Though the unlabeled protein is generally not visible in isotope-filtered NMR experiments, it results in an effective reduction in yield of the observable sample. By altering the cSPP system and separating the induction of the MazF toxin, required to convert cells into a semiquiescent state prior to condensation, from the expression of the target gene, we are now able to eliminate the unlabeled protein fraction and improve the isotope incorporation. Here we describe a series of pCold(tet) vectors with various features that can be used in the dual inducible cSPP(tet) system to obtain high-quality isotopically enriched protein at as little as 2.5% the cost of traditional methods.
KeywordsAnhydrotetracycline Isotope labeling MuLV IN pCold vectors Structural genomics Triple resonance NMR
Condensed single protein production
- M-MuLV IN
Moloney Murine Leukemia Virus Integrase
Translation enhancing element
We thank M. Suzuki, L. Mao, Y. Tang, and P. Rossi for helpful discussions in the course of this work, and for their comments on the manuscript. This work was supported by the National Institutes Health Grants RO1 GM070837 (to M.J.R.), U54 GM074958 (G.T.M. and M.I.), U54 GM75026 (G.T.M. and M.I.), 1R01 GM085449 (M.I.). W.M.S. was supported by NIH training grants T32 GM08360 and T32 A1007403.
- 5.Maloy SR, Stewart VJ, Taylor RK (1996) Genetic analysis of pathogenic bacteria: a laboratory manual. Cold Spring Harbor Laboratory Press, NYGoogle Scholar