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This protein is thought to make an early contribution to the assembly process through delivery of ��-tubulin onto a second protein, TBCD. In one model for microtubule biogenesis this TBCD�C��-tubulin complex then facilitates ��/��-tubulin heterodimer formation (Lundin et al., 2010 ?). We describe the high-resolution crystallographic analysis of L. major TBCA (LmTBCA) and provide comparisons with orthologous structures derived from yeast, plant and mammalian sources. Conserved features suggest several structural factors that might be important for interactions between TBCA and a cognate tubulin and provide a model to help formulate hypotheses and further dissect a key aspect of eukaryotic biology. 2.?Materials and methods ? 2.1. Cloning, expression and purification of recombinant protein ? The gene predicted to encode TBCA from L. major Friedlin in GeneDB (LmjF.32.2970; Logan-Klumpler Y-27632 concentration et al., 2012 ?) was amplified by PCR from genomic DNA and subcloned into the expression vector pET-15b-TEV (modified from pET-15b, Novagen). Under the control of the T7 promoter, this vector produces protein with an N-terminal hexahistidine tag that is cleavable by tobacco etch virus (TEV) protease. The resulting plasmid was transformed into Escherichia coli strains BL21 (DE3) Gold and B834 (DE3) for the production of native and selenomethionine bepotastine (SeMet)-derived proteins, respectively. Typically, bacteria were cultured at 310?K to an optical density at 600?nm of 0.5�C0.6 in Luria�CBertani medium containing 50?mg?l?1 carbenicillin or in methionine-deficient medium supplemented with 50?mg?l?1 carbenicillin and 40?mg?l?1 selenomethionine (Molecular Dimensions). Protein production was then induced with 1?mM isopropyl ��-d-1-thiogalactopyranoside and growth continued for approximately 16?h at 295?K prior to harvesting the cells by centrifugation (3500g for 30?min at 277?K). The cell mass was stored frozen at 253?K until required. The cells were thawed on ice and then lysed using a French press at 110?MPa, and the lysate was clarified Olaparib in vivo by centrifugation at 37?500g for 30?min at 277?K. The soluble supernatant was filtered (0.2??m) and loaded onto a 5?ml HisTrap HP column (GE Healthcare) for an initial affinity-chromatography capture step. The product was then eluted between 150 and 180?mM imidazole; fractions containing LmTBCA were pooled and treated with TEV protease at 303?K for 2?h. Dialysis at room temperature to remove excess imidazole was followed by reverse affinity chromatography prior to a final purification step by size-exclusion chromatography using a calibrated Superdex 75 26/60 gel-filtration column in 50?mM Tris�CHCl, 250?mM NaCl pH 7.5. In all purifications that were carried out, the gel-filtration trace of LmTBCA indicated the presence of two species in an approximate ratio of 2.5:1 with approximate molecular weights of 27 and 43?kDa, respectively. The theoretical molecular weight is about 14?kDa.
