Bacterial expression and isotope labeling of AIMP 1 / p 43 codosome protein for structural studies by multidimensional NMR spectroscopy

N. V. Vorobyova1, 2, D. M. Lozhko1, I. Yu. Zhukov3, 4, A. I. Kornelyuk1, 2 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, Poznan, Poland, 61-614 vorobyova_natali_0307@ukr.net


Introduction
In mammals, nine aminoacyl-tRNA synthetases are as sociated with three auxiliary proteins AIMP1/p43, AIMP2/p38 and AIMP3/p18 (aminoacyl tRNA synthetase interacting with multifunctional proteins, AIMPs) to form a stable multiprotein complex composed of two subcomplexes [1].One subcomplex is composed of arginyl-tRNA synthetase, glutaminyl-tRNA synthetase and AIMP1/p43; another subcom-plex includes the rest of the components.These two subcomplexes are connected via AIMP2/p38.The formation of interaction between AIMP1/p43 and ArgRS-GlnRS is one of the key stages of assembly of the full complex [2].The AIMP1/p43 component, which has a potent tRNA binding capacity is associated to the complex via its N-terminal moiety [3].
At the present stage of the development of medicine, the usage of recombinant proteins as therapeutic agents becomes widespread.Protein drugs offer many new therapeutic approaches mainly aimed at the treatment of severe chronic diseases and cancer.The main problem of these drugs is their instability.To overcome this problem in pharmacology, additional chemicals such as cyclodextrins [4] can be used, which as auxiliary agents are capable to reduce the level of aggregation of the protein components, to enhance the resistance to blood proteolytic enzymes and increase their solubility.
The high-resolution 3D structure of full-length AIMP1/p43 has not been established yet in either monomer or dimer state.The goal of the present work is the optimization of protocols of expression and purifi cation of AIMP1/p43 in Escherichia coli bacteria to achieve maximal quantity of the uniformly 15 N or / and 13 C, 15 N isotope-labeled recombinant protein.The prepared sample was used to acquire preliminary heteronuclear NMR spectra and to inspect the dispersion of resonances.Additionally, we controlled the experimental conditions (buffer, temperature, pH) from the point of view of long-term protein stability in solution, requested to record the multidimensional NMR data sets.This study is an initial step to evaluate the high-resolution 3D AIMP1/ p43 structure using modern techniques recently developed in multidimensional NMR spectroscopy.
M9 minimal medium (1 L): 100 ml of 10 x M9 salt; 1 g of [ 15 N]-NH 3 Cl; 10 ml of 100 х of trace metal solution; 1 ml of 2 M MgSO 4 ; 100 μl 1 M CaCl 2 ; 2 g of glucose; 1 ml of 1 mg/ml biotin; 1 ml of 1 mg /ml thiamine; 1 ml of 30 mg /ml kanamycin.Agar (1.5%) M9 minimal medium was prepared with the addition of 1.5 g of agar per 100 ml of liquid medium.M9 salts ( Recombinant protein AIMP1/p43 was obtained from 400 ml of bacterial suspension on M9 minimal medium with 15 NH 4 Cl and 30 μg /ml kanamycin.The culture was grown at 37 C on a thermostatted shaker (Environmental Shaker Incubator ES-20) to an optical density of A 600 = 0.511 and synthesis of target protein was induced by adding 1 mM IPTG, followed by incubation for 4.0 h at 30 C.The cells were harvested by centrifugation at 4000g for 20 min (centrifuge K-23) and frozen at -20 C overnight.The frozen cell pellet was suspended in 48 ml of cell lysis buffer (50 mM sodium phosphate buffer, pH 8.0, 500 mM NaCl, 10 mM imidazole, 5 mM β-mercaptoethanol, 5 mM DTT, 5 % glycerol, 1 mM lysozyme, 1 mM EDTA, 1 mM PMSF).Ultrasonic lysis of cells was carried out (6 cycles of 20 s, 20 s intervals).The sonicated cells were clarifi ed by centrifugation at 13,000 rpm for 30 min to micro-centrifuge Sigma 1-13.

Affi nity chromatography on Ni-NTA-agarose column
The supernatant was applied to the equilibrated Ni-NTA-agarose column, washed with 20 ml of wash buffer -50 mM sodium phosphate buffer, pH 8.0, 500 mM NaCl, 20 mM imidazole, 5 mM β-mer captoethanol.The target protein was eluted with 5 ml of elution buffer -50 mM sodium phosphate buffer, pH 8.0, 150 mM NaCl, 200 mM imidazole, 5 mM β-mercaptoethanol -and collected in 6 fractions (each one had volume of 1 ml) stored at 4 C.Protein concentration was determined by UV absorption and by the method of Bradford [13].The fi nal concentration of protein was performed on micro concentrators («Millipore», France) to the desired concentration.The purity of the obtained protein p43 was checked by electrophoresis under denaturing conditions in the presence of sodium dodecyl sulfate 12 % polyacrylamide gel for Lemmli [14] using a mixture of marker proteins («Thermo Scientifi c», Lithuania).

Spectrophotometric determination of protein concentration
UV-absorption spectra of recombinant AIMP1/p43 were measured using a spectrophotometer BioMate-5 («Termo Scientifi c», UK) in quartz cuvettes with an optical path length of 1 cm.Protein concentration was determined spectrophotometrically using absorption at 280 nm (A 280 ) based on extinction coeffi cient e AIMP1/p43 = 10220 M -1 cm -1 .

NMR spectroscopy
All NMR experiments were carried out at 20 C in the NMR spectrometer Agilent DDR2 800 ( 1 H reso-nance frequency is 800 MHz), equipped with four frequency channels, 1 H/ 13 C/ 15 N probehead with inverse detection and z-gradient unit.The 1 H and 15 N dimensions were referenced in respect to external sodium 2,2-dimethyl-2-silapentane-5-sulfonat (DSS) using previously described procedure [20].All NMR spectra were processed using NMRPipe [15] and analyzed with Sparky [16] software.

Results and Discussion
Determination of the spatial structure of the AIMP1/ p43 protein in solution at physiological conditions is an urgent task.At the moment only C-terminal fragment of 3D structure (EMAP II domain) has been determined.According to our bioinformatics analysis, the AIMP1/p43 protein contains a long structurally disordered fragment in the middle of polypeptide (unpublished results).In such case, NMR spectroscopy is the only method which allows determining the 3D structure of proteins in solution and establishing the correlation between crystal and solution structures.It should be noted that the NMR spectroscopy is the only method for structure determination of intrinsically disordered proteins.
Usually, NMR spectroscopy determination of the protein 3D structure in solution includes the isotope labeling procedures in the following combina-  15 N/ 13 C double labeling and 2 H/ 13 C/ 15 N triple labeling.At the fi rst step, a studied protein is labeled with 15 N isotope in order to verify the feasibility of the next stages of the research.To determine the solution structure, we performed the bacterial expression and 15 N isotope labeling of the recombinant AIMP1/p43 polypeptide according to the technique described earlier [17].The optimized protocol of the synthesis and purifi cation procedures fi nally yields about 8 mg of recombinant uniformly 15 N-labeled AIMP1/p43 protein from 1 L of cell culture of E. coli, which is economically viable to apply the method of isotopic labeling.In this case, the protein is suffi ciently soluble and relatively for 4-7 days stable, as determined from the NMR spectra.An analysis of the AIMP1/p43 protein by gel electrophoresis under denaturing conditions showed its high homogeneity and purity (at least 95 %) (Fig. 1).
A signifi cant yield of the protein indicates the absence of such problems as the protein expression toxicity for E. coli cells, instability, improper processing and ineffi cient translation of the protein.The obtaining of the AIMP1/p43 protein preparations at concentrations of 0.3-0.5 mM is well maintained by the intensive dialysis against 50 mM sodium phosphate buffer, 250 mM NaCl, 1 mM DDT at pH 8.0 which was performed for the removal of imidazole and organic contaminants.
Two-dimensional 1 H-15 N HSQC spectra of the 15 Nlabeled AIMP1/p43 protein demonstrate good dispersion of resonances coming from amide groups of the polypeptide chain (Figure 2).A comparison with the previously recorded data for the EMAPII protein which constitutes the C-terminal module of AIMP1/ p43 exhibited good correlation.This strongly supports an idea that the 3D structure of the C-terminal part of the AIMP1/p43 protein is very similar to the previously determined 3D structure of the EMAP II protein [18].The presented NMR data for the AIMP1/p43 protein indicated also the presence of some additional signals in comparison to the EMAP Bacterial expression and isotope labeling of AIMP1/p43 codosome protein II spectrum (Fig. 2).The additional signals detected are well separated from the signals coming from the C-terminal part, are characterized by high amplitude and small linewidth, and more probably correspond to the amino acids located in the long unstructured part of the AIMP1/p43 structure.In this regard it is important to note that our prediction of the AIMP1/p43 secondary structure suggested the presence of α-he lical fragment at the N-terminal part connected with the Cterminal domain by the long mobile loop (unpublished data).Additionally, these data correlate well with the results of X-ray diffraction studies of the ArgRS-Gl-nRS-AIMP1 complex structure [2].However, so far these additional NMR signals have not been exactly assigned to the specifi c amino acid residues of AIMP1/ p43 and will be the subject of our further research.
Additionally, NMR spectroscopy may also be useful to explore the interactions of the protein with different ligands and to determine the structure of these complexes.It is known that AIMP1/p43 revealed some cytokine properties, which may be modulated in some nanocomposite complexes.In this regard, we focused on creating some nanocomposite systems of AIMP1/p43 for further implementation as novel potential anticancer drugs.NMR spectroscopy will be very useful to determine the structure of different nanocomposite complexes of AIMP1/p43 for biomedical application.
The obtained data confi rm the existence of a stable NMR spatial structure of the p43 protein in aqueous solution and the possible formation of α-helical fragments in the N-terminal part of the polypeptide.The results suggest the possibility of determining the AIMP1/p43 protein 3 D structure in solution by multi-dimensional NMR spectroscopy.

Fig. 2 .
Fig. 2. Two-dimensional NMR spectrum of 1 H/ 15 N HSQC (heteronuclear single quantum correlation experiment) AIMP1/p43 recombinant protein in solution, which records the signals of amide groups of the polypeptide core protein, recorded at a frequency of 800 MHz by NMR spectrometer at Agilent DDR2 800.Assigned signals belongs to the C-terminal part of protein (EMAP II), unassigned (marked by red) -N-terminal part