Cloning, expression and purification of D-Tyr-tRNATyr-deacylase from Thermus thermophilus
Biopolymers and Cell
D-Tyr-tRNA Tyr -deacylase (DTD) is a conservative enzyme, found in all domains of life, which ensures an additional checkpoint in the recycling of misaminoacylated D-Tyr-tRNA Tyr . DTD is capable of accelerating the hydrolysis of the ester linkage of D-Tyr-tRNA Tyr producing a free tRNA and D-tyrosine, thereby preventing an incorrect incorporation of D-amino acids into proteins. Deacylase distinguishes between D-and 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-tRNA Tyrdeacylase 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. K e y w o r d s: D-amino acids, D-Tyr-tRNA Tyr -deacylase from T. thermophilus, cloning, expression, purifi cation.