CLONING AND CRYSTALLOGRAPHIC ANALYSIS OF HUMAN CARBONIC-ANHYDRASE-I

The cDNA encoding the human carbonic anhydrase I (hCAI) gene was prepa red using a specific primer and total mRNA extracted from human erythr oleukaemia (HEL) cell line. The cDNA was amplified by PCR and was clon ed into the expression vector pET-3a. Nucleotide sequencing of the clo ned gene showed the following amino acid changes: Val31Ile and Val218A la, when compared to erythrocyte hCAI sequence. The gene was induced b y IPTG, and the protein purified by affinity column chromatography was found to be as active as erythrocyte human carbonic anhydrase I (eHCA I). N-terminal amino acid sequencing of the purified protein revealed that two methionines at the N-terminus have not been cleaved off by pr ocessing in E. coli. The recombinant human carbonic anhydrase I (rHCAI ) has been crystallized for the first time. These crystals belong to t he trigonal space group P3(1)2 1 with unit cell dimensions of a = b = 120.2 Angstrom, and c = 88.8 Angstrom. The crystals contain two molecu les in their asymmetric unit, Diffraction data to 2.2 Angstrom resolut ion has been collected using an imaging plate diffractometer. The stru cture has been determined by the molecular replacement method. The ove rall folding of the rHCAI is similar to that of native eHCAI, the RMSD for the superposition of 254 C-alpha atom pairs being 0.54 Angstrom. In contrast to orthorhombic crystals of eHCAI, crystals of rHCAI are e xtremely sensitive to X-rays.