Biopolym. Cell. 2015; 31(2):109-114.
http://dx.doi.org/10.7124/bc.0008D4
Structure and Function of Biopolymers
Bacterial expression and isotope labeling of AIMP1/p43 codosome protein for structural studies by multidimensional NMR spectroscopy
1, 2Vorobyova N. V., 1, 2Lozhko D. N., 3, 4Zhukov I. Yu., 1, 2Kornelyuk A. I.
  1. Institute of Molecular Biology and Genetics, NAS of Ukraine
    150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680
  2. Institute of High Technologies,
    Taras Shevchenko National University of Kyiv
    2, korp.5, Pr. Akademika Hlushkova, Kyiv, Ukraine, 03022
  3. Institute of Biochemistry and Biophysics, Polish Academy of Sciences
    5a, Pawinskiego, Warsaw, Poland, 02-106
  4. NanoBioMedical Centre, Adam Mickiewicz University
    85, Umultowska Str., 61-614 Poznan, Poland

Abstract

AIMP1/p43 protein is a structural component of multisynthetase complex (codosome) in eukaryotes, which reveals both tRNA binding and cytokine activities. Aim. Bacterial expression and purification of isotopically-labeled recombinant AIMP1/p43 protein in E. coli cells for studying its solution structure by multidimensional NMR spectroscopy. Methods. AIMP1/p43 protein was expressed in E. coli BL21(DE3)pLysE cells on M9 minimal medium with 15N isotope labeling and purified by metal-chelated chromatography. Heteronuclear 2D 1H-15N NMR experiments were performed in solution at 293 K on Agilent DDR2 800 NMR spectrometer. Results. The AIMP1/p43 protein was obtained in uniformly 15N-labeled form as an NMR sample. A high dispersion of resonance signals in the 2D 1H-15N HSQC NMR spectra confirmed the presence of its compact 3D protein structure. The NMR spectrum of AIMP1/p43 demonstrated a high signal-to-noise ratio and sufficient stability to acquire other multidimensional NMR data sets for determination of the structure of AIMP1/p43 protein in solution. Conclusions. The 15N-labeled AIMP1/p43 protein was stable for 4–7 days, which makes possible acquiring the critical NMR experimental data for detailed structural analysis in solution. Our data on the initial NMR spectra indicated the presence of some additional signals in comparison with the NMR spectrum of EMAP II which could be assigned to amino acids of the N-terminal α-helical fragment of AIMP1/p43.
Keywords: cytokine, AIMP1/p43, protein expression, isotope labeling, NMR-spectroscopy
Full text: (PDF, in English)

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