Biopolymers and Cell. 2011; 27(4): 273-278
BACTERIAL EXPRESSION AND 13C/15N ISOTOPIC LABELING OF EMAPII CYTOKINE FOR STRUCTURAL STUDIES BY NMR SPECTROSCOPY
Lozhko D. M.,1 Zhukov I. Yu., Kornelyuk A. I.
Institute of Molecular Biology and Genetics, NAS of Ukraine
150, Akademika Zabolotnogo Str., Kyiv, Ukraine, 03680
1 Institute of Biochemistry and Biophysics, Polish Academy of Sciences
5a, Pawinskiego, 02-106 Warsaw, Poland
Aim. Bacterial expression and isotopic labeling of recombinant EMAP II in preparative scale with the aim to study its spatial structure by NMR spectroscopy. Methods. The recombinant 15N, 13N-labeled EMAP II protein was expressed in Escherichia coli BL21(DE3) pLysE on M9 minimal medium and purified by metal-chelated chromatography on Ni-NTA agarose. Quality of obtained for NMR studies N and 15N/13C-double labeled EMAP II was confirmed by mass spectroscopy and used for preparation of NMR samples. Results. The preparations of 15N and 15N/13C double labelled EMAP II were obtained. The recorded NMR spectra of EMAP II were analyzed. Conclusions. Dispersion of NMR signals in two-dimensional 1H/13C/15N and NHSQC spectra confirm the presence of compact three-dimensional structure of the protein. The NMR samples were stable at least for 5–6 days at chosen conditions. These results will be used as a starting point for determination of high resolution three-dimensional structure of recombinant EMAP II cytokine in solution by NMR spectroscopy.
Keywords: cytokine, EMAP II, isotopic labeling, mass spectroscopy, NMR spectroscopy, two-dimensional spectra.