Cloning, expression and purifi cation of D-Tyr-tRNATyr-deacylase from Thermus thermophilus

D-Tyr-tRNATyr-deacylase (DTD) is a conservative enzyme, found in all domains of life, which ensures an additional checkpoint in the recycling of misaminoacylated D-Tyr-tRNATyr. DTD is capable of accelerating the hydrolysis of the ester linkage of D-Tyr-tRNATyr producing a free tRNA and D-tyrosine, thereby preventing an incorrect incorporation of D-amino acids into proteins. Deacylase distinguishes between Dand Laminoacyl moieties and does not hydrolyze L-aminoacylated tRNA. The structural bases of this specifi city and the mechanism of D-aminoacyl-tRNA hydrolysis are poorly understood. Aim. To clone D-Tyr-tRNATyrdeacylase from T. thermophilus (DTDTT), optimize the conditions for its expression in E.coli and develop an effi cient purifi cation procedure yielding the high quality enzyme suitable for the structural and functional studies. Methods. For amplifi cation of DTD gene from T. thermophilus genomic DNA and its cloning into the pProEXHTb expression vector modern techniques were applied. Purifi cation of the recombinant DTD protein was done with three types of column chromatography. His-tag was cleaved out from DTD by TEV protease. The cleavage was confi rmed by Western blot analysis with anti-His-tag antibodies. Molecular weight of purifi ed DTDTT was determined by the gel-fi ltration. Results. The expression construct pProEXHTb, containing DTD sequence from T. thermophilus, was obtained and successfully expressed in the BL21(DE3)pLysS E.coli strain. The protein of interest was purifi ed to homogeneity by the combination of affi nity (Ni-NTA), anion-exchange (Q-Sepharose) and size-exclusion (Superdex S 200) chromatographies. 2 mg of more than 90% pure recombinant DTD can be obtained from 1 L of bacterial culture. Molecular weight of purifi ed DTD from T. thermophilus was determined to be 32 kDa, suggesting its dimeric structure. Conclusions. The pProEXHTb expression vector can be used for expression of DTD from T. thermophilus. The preparative amounts of DTD can be obtained after the three-step chromatographic procedures and used for further functional and structural studies.


Introduction
D-amino acids are present in the cells of various species from bacteria to mammals. In the bacterial walls D-amino acids contribute to the resistance to proteolytic digestion [1]. They also could be considered as bacterial growth inhibitory factors that prevent a biofi lm formation [2]. Furthermore, D-amino acids have also been found in the proteins extracted from aged human tissues [3]. These are the myelin basic pro-tein, erythrocyte proteins, and L-amyloid pepti des from Alzheimer disease brains [4]. D-amino acids are shown to have toxic effects on the cells in both prokaryotes and eukaryotes [5,6,7,8].
Aminoacyl-tRNA-synthetases (aaRS), being specifi c to L-amino acids, ensure the fi rst step of D-amino acids' exclusion from protein synthesis. However, the stereospecifi city of these enzymes is not absolute: several aaRS have been found to charge tRNAs with D-amino acids [9,10]. D-Tyr-tRNA Tyr -deacyla-se is an enzyme responsible for the recycling of misaminoacylated D-Tyr-tRNA Tyr , hydrolyzing an ester bond between the amino acid and tRNA. However, this enzyme has a broad specifi city [11], and may accommodate different D-aminoacyl moieties, for example, D-Tyr, D-Trp, D-Asp and D-Phe [9,10].
The fi rst observations of the deacylase editing activity in the extracts of E.coli, S.cerevisiae, rabbit reticulocytes and rat liver were reported by Calendar and Berg [9]. Later Soutourina et al. purifi ed D-Tyr-tRNA Tyr -deacylase from E.coli [10,12] and S.cerevisiae [10,13]. Plant DTD was discovered as a product of GEK1 gene that is involved in the ethanol tolerance in Arabidopsis thaliana [14]. The identifi cation of DTD in other groups of organisms, including human [15], confi rms its widespread distribution in all kingdoms of life and may be considered as an important checkpoint of the translation machine specifi city. In addition, the DTD amino acid sequences identity among prokaryotes and eukaryotes are highly conservative [16,17], suggesting a high conservative function of this enzyme in all living organisms.
Three classes of deacylases have been identifi ed: class DTD1 has been found in most bacteria and all eukaryotes [12], class DTD2 has been discovered in archea and plants [14,18], class DTD3 -in most cyanobacteria [11]. The species with DTD1 have the yihZ and dtd orthologous genes, responsible for the deacylase activity. Despite the fact that the homologues of dtd were found in different pro-and eukaryotic genomes, another type of DTD (dtd2) was identifi ed in archea and subsequently in plants. In contrast to the mainly dimeric DTD1 proteins, DTD2 has a monomer structure. In addition, the activity of deacylases from the second class depends on the presence of Zn 2+ ions. The third type of D-Tyr-tRNA Tyrdeacylases has been reported to be encoded by the dtd3 gene (homologous to dtd1). DTD3 is a metalenzyme with two active sites for metal ions binding: the fi rst one binds only Zn 2+ , the second -Ni 2+ , Mn 2+ and Co 2+ ions.
Some functional investigations of E.coli [12], S. cerevisiae [10,13], archaeal [18] D-Tyr-tRNA Tyrdeacylases were performed, but profound structural research that may explain the mechanism of D-aa-tRNA hydrolysis by DTD, is still to be carried out. Unfortunately, the proposed catalytic mechanism of DTD based on the crystal structures available at this time is controversial and remains to be clarifi ed [17,19].
In order to investigate the structural and functional properties of deacylase, one needs to have the preparative quantities of this enzyme of a high purity. In this work we describe the expression and purifi cation of DTD from T. thermophilus (DTDTT).
DTDTT purifi cation by affi nity, anion exchange and size-exclusion chromatography. Pre-culture of E. coli BL21(DE3)pLysS cells (50 ml), carrying the recombinant plasmids, was grown overnight at 37 C in Terrifi c-Broth with appropriate antibiotics. The cu lture (2.5 L) was inoculated with pre-culture in dilution 1:100 and grown at 37 C on TB medium supplemented with ampicillin (100 μg/ml) and chloramphenicol (35 μg/ml). The expression of DTDTT was induced by the addition of 0.6 mM IPTG at A 600 = 0.6 and the culture growth continued for 4 h at 37 C.
The cells were harvested by the centrifugation for 15 min at 6000 × g (4 C). The bacterial cell pellet was resuspended in 70 ml of 25mM Tris-HCl (pH 7.5), 1mM PMSF, 10 mM β-mercaptoethanol supplemented with 1.5 tablets of EDTA free protease inhibitors cocktail. The cells were incubated on ice for 30 min and then disrupted by sonication 8 × 30 sec with 1 min breaks (4 C). All subsequent steps were conducted at 4 C. The cell debris was precipitated by centrifugation at 20 000 × g. The clear supernatant was recovered and concentrations of sodium chloride and imidazole («Sigma», USA) were adjusted to 300 mM and 10 mM, respectively. The obtained solution was mixed with Ni-NTA Sepharose Fast Flow resin (5 ml of 50 % slurry, «GE He althcare», Sweden), pre-equilibrated with the same buffer, and incubated for 1.5 h on the rotor shaker at 130 rpm. The resin was washed with buffer A (25 mM Tris-HCl, pH 7.5, 0.1 mM PMSF, 1 mM β-mer cap toethanol, 300 mM NaCl, 10 mM imidazole) and then with buffer A containing 600 mM NaCl. DTDTT was eluted from the column by 400 mM imidazole in buffer A. The collected fractions were analyzed by SDS-PAGE. The purest fractions were combined and dialyzed overnight against an appropriate buffer for TEV protease digestion -buffer B (50 mM Tris-HCl (pH 7.5), 0.1 mM PMSF, 1 mM DTT, 0.5 mM ED TA). After the dialysis His-tag-residues were cut off from DTDTT by recombinant TEV protease as follows: 1 A 280 of TEV per 5 A 280 of DTD during overnight digestion at 4 °C [22]. The resulting solution from the fi st purifi cation step was diluted to 1 A 280 unites/ml and applied on Q-Sepharose Fast Flow column («Pharmacia», Sweden) (1.35 x 4 cm, V = 6 ml), preequilibrated by buffer B. A column was washed by the same buffer. The elution was performed at a fl ow rate of 0.6 ml/min with a linear gradient of 200-800 mM NaCl (70 ml).
The protein-containing fractions were detected by Bradford assay, the DTD-containing fractions we re analyzed by 15 % SDS-PAGE. The collected frac tions containing DTDTT were dialyzed overnight against 25 mM Tris-HCl (pH 7.5), 1 mM DTT at 4 C and concentrated on 10 kDa Centricon («Merck», Ger-

Western blot analysis of DTDTT before and after TEV protease treatment
The proteins were separated in 15% SDS-PAGE and transferred onto a prepared 0.45 μm polyvinyl difluoride (PVDF) membrane (incubated for 1 min with MeOH and rinsed once by Towbin buffer («Bio-Rad», USA)) on Trans-Blot Semi-Dry electrophoretic transfer system («Bio-Rad», USA). The membrane was blocked overnight by 5 % non-fat milk in PBST buffer solution (PBS plus 0.5 % Tween-20). After blocking, membrane was incubated with mouse anti-His mono-clonal antibodies («Sigma», USA) in dilution 1 : 6000 for 1 h at room temperature. Then, the membrane was extensively washed by PBST buf fer (4 times × 5 min) and treated with secondary anti-mouse antibodies (Jackson Immuno Research Inc., USA), conjugated to peroxidase, at 1 : 10000 working dilution for 1 h. After this incubation the extensive (4 times × 5 min) washing with PBST was performed. The immune complexes were detected by ECL detection kit (EMD Millipore Immobilon Western Chemiluminescent HRP Substrate) («Milipore», USA) using X-ray fi lm.

Results and Discussion
Creation of DTD expressing construction and expression of recombinant protein in different media. Previously, we tried to express the DTD gene from T. thermophilus in pET15b, pET28b and pET29b vectors (under control of T7-promotor and lac-operator), but it resulted in low expression level of the target protein even after 24 h of IPTG induction. To overcome this problem we decided to switch to pProEXHTb expression vector, which possess' Trc promoter. pProEXHTb was earlier shown to produce large quantities of the target proteins, during a short time IPTG induction [24].
The expression level of DTDTT in E. coli BL21 (DE3)pLysS cells was checked under varied IPTG concentrations and in several media (LB, TB, P, 2xTY). The best conditions obtained were as follows: 4-5 h of 0.6 mM IPTG induction at 37 C in Terrifi c Broth medium (Fig. 1). These conditions were further used for the preparative DTDTT expression. many) at 5500 rpm to 4.54 A 280 unites/ml (≈ 12 mg/ ml).
The eluted fractions were col lected and analyzed by 15 % SDS-PAGE. The de acylase containing fractions were combined and con centrated to 8 mg/ml. The enzyme was supplemented by 50 % glycerol and stored at -20 C.

Analytical gel fi ltration of proteins
To determine the approximate molecular weight of DTDTT the gel fi ltration on Hi-Load 16/60 Superdex S 200 (150 ml, «Pharmacia Biotech») was used. The column was pre-equilibrated with 25 mM Tris-HCl (pH 7.5), 1 mM DTT, 150 mM NaCl, 0.003 % NaN 3 . All samples were run at 1 ml/min fl ow rate. The void column volume (V o ) was determined by blue dextran (2 MDa). A set of proteins were used for the column calibration: ferritin (450 kDa), catalase (240 kDa), β-amilase (200kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carboanhidrase (29 kDa), cytochrome c (12,4 kDa). The molecular weight of DTDTT was determined by a comparison of its V e / V o index with those of the known protein standards. The logarithms of the molecular weights of marker proteins were plotted against their appropriate ratios of the elution volume to the column void volume (V e / V o ). Calibration curve is shown in Fig. 6.

Purifi cation of His-DTDTT
The fi rst step of the His-DTDTT purifi cation was the affi nity chromatography on Ni-NTA. The result is presented in Fig. 2. The enzyme, eluted from the column by 400 mM imidazole, contained the contaminations of higher molecular weight proteins. Washing the column with a buffer supplemented with 20 mM imidazole slightly increased the DTDTT purity but decreased its yield. In addition, washing the column with 1 M NaCl did not signifi cantly diminish the amount of impurities (data not shown). Unfortunately, we could not improve the quality of the DTDTT preparation after this step of purifi cation.    It is worth noting that the ratio A 260 /A 280 of DT-DTT after the affi nity purifi cation step and His-tag cleava-ge was about 1.0 that may refl ect the presence of nuc-leic acid contaminations. To remove the nucleic acid fragments we applied an anionexchange chromatography. Unfortunately, during this step we could not get rid of the protein contamination present in the DTDTT preparation. We applied various linear gradients (from 0 to 1M NaCl and from 50 mM to 800 mM NaCl), but this did not improve the quality of DTDTT. Finally, we used the gradient from 200 to 800 mM of NaCl, which allowed us to remove the nucleic acids and some protein contaminations. After Q-Sepharose column, DTDTT had typical absorbance ratio A 260 /A 280 = 0.5-0.6. SDS-PAGE of fractions obtained after Q-Sepharose is presented in Fig. 4.
To get rid of the higher molecular weight impurities in the DTDTT preparation we performed a size-exclusion chromatography as a fi nal step of the purifi cation procedure. The elution profi le of DTDTT from the column is shown in Fig. 5, B. As can be judged from the elution profi le, DTDTT was effi ciently separated from the contaminating proteins and eluted from the column as a single peak (Fig. 5, A). According to SDS-PAGE ( Fig. 5, B) the purity of DTDTT may be more than 90 %.

Molecular weight determination of D-Tyr-tRNA Tyr -deacylase
The molecular weight of the D-Tyr-tRNA Tyr deacylase was deduced from a comparison of its elution time on Superdex S 200 column with the proteins of known molecular weight. Column was calibrated as described in «Materials and methods». The elution volume (V e ) of DTDTT from Superdex S 200 was determined to be 11 ml. V e / V o index of DTDTT was calculated to be 1,888. According to the calibration curve (Fig. 6) the molecular weight of DTDTT was estimate to be 32 kDa. Based on the amino acid sequence of DTDTT (152 a. a. residues), its theoretical molecular weight is 16.7 kDa. Thus, the purifi ed recombinant DTDTT most probably is a dimer in solution. However, a monomeric unglobular form of this protein with an elongated shape could not be excluded.

Conclusions
The cDNA encoding D-Tyr-tRNA Tyr -deacylase from T. thermophilus was cloned into pProEXHTb vector and successfully expressed in BL21(DE3)pLysS strain in TB medium. The purifi cation procedure described here allows obtaining 2 mg of the pure enzyme from 1 L of the bacterial culture. According to the gel fi ltration analysis recombinant DTDTT may exist as a dimer in solution. The obtained protein will be used for further structural and functional studies.