Bacterial expression and isotope labeling of AIMP1/p43 codosome protein for structural studies by multidimensional NMR spectroscopy

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 purifi cation 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 confi rmed 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.