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Overexpression, purification, crystallization and structure determination of AspB, a putative aspartate aminotransferase from Mycobacterium tuberculosis
Author(s) -
Saroj Deepak Chandra,
Singh Khundrakpam Herojit,
Anant Avishek,
Biswal Bichitra K.
Publication year - 2014
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 37
ISSN - 2053-230X
DOI - 10.1107/s2053230x14011820
Subject(s) - orthorhombic crystal system , crystallization , recombinant dna , mycobacterium tuberculosis , chemistry , monoclinic crystal system , molecular replacement , microbiology and biotechnology , crystal structure , biology , biochemistry , crystallography , tuberculosis , gene , medicine , organic chemistry , pathology
A recombinant version of a putative aspartate aminotransferase, AspB (encoded by the ORF Rv3565), from Mycobacterium tuberculosis ( Mtb ) was overexpressed in M. smegmatis and purified to homogeneity using liquid chromatography. Crystals of AspB were grown in a condition consisting of 0.2 M ammonium phosphate monobasic, 0.1 M calcium chloride dihydrate employing the hanging‐drop vapour‐diffusion method at 298 K. The crystals diffracted to a limit of 2.50 Å resolution and belonged to the orthorhombic space group P 2 1 2 1 2 1 , with unit‐cell parameters a = 93.27, b = 98.19, c = 198.70 Å. The structure of AspB was solved by the molecular‐replacement method using a putative aminotransferase from Silicibacter pomeroyi (PDB entry 3h14 ) as the search model. The template shares 46% amino‐acid sequence identity with Mtb AspB. The crystal asymmetric unit contains four AspB molecules (the M r of each is 42 035 Da).