Gsat And Wtap Protein Cloning, Expression, Purification And Preliminary Crystallographic Analysis

1 .Cloning, expressing, purification, crystallization and preliminary X-ray diffraction analysis of the glutamate -1-semialdehyde aminotransferase from Bacillus subtilis5-aminolevulinic acid (ALA) is the first committed universal precursor in the tetrapyrrole biosynthesis pathway. Plants, algae and many other bacteria synthesize ALA from glutamate by a C5 pathway in which the carbon skeleton of glutamate is converted into ALA by a series of enzymes. Glutamate-1-semialdehyde aminotransferase (GSAT) is the last enzyme in this pathway. The gene that codes for GSAT was amplified from cDNA library of B. subtilis and overexpressed in Escherichia coli strain BL21 (DE3). The protein was about 95% pure after purified by Ni-NTA column (Amersham Biociences) and crystallized. Well diffracting single crystals were obtained by the vapor-diffusion method in hanging drops. Preliminary X-ray diffraction studies yielded excellent diffraction data to a resolution of 2.0 A. The crystals have the symmetry of the monoclinic space group C2 with unit-cell parameters a=111.428, b=56.496, c=81.028 A, 3=131.928°. Assuming the presence of one molecule per asymmetric unit, the Matthews coefficient (V_M) was calculated to be 2.0 A~3/Da, which corresponds to 39.4% solvent content. Determination the structure of GSAT by molecular replacement method using Glutamate-1-semialdehyde aminotransferase from Synechococcus (3GSB) as a search model (58% amino-acid sequence identity) are underway.2. Cloning, Expression, Purification and the structural research of WTAPThe Wilms' tumour suppressor gene is essential for the normal development of...