Crystallization and preliminary X-ray analysis of insect antifreeze protein from the beetle Tenebrio molitor.

Hyperactive antifreeze protein from the beetle Tenebrio molitor (TmAFP) was produced in Escherichia coli and purified by gel-permeation chromatography and HPLC. An iodinated derivative was prepared by incubating the 8.5 kDa TmAFP with N-iodosuccinimide. Native and iodinated TmAFP produced two different crystal forms when crystallized using the hanging-drop vapor-diffusion technique. Native crystals were rectangular plates that diffracted to approximately 2.5 A resolution. They were monoclinic and belonged to the space group P2(1), with unit-cell dimensions a = 38.4, b = 73.4, c = 59.3 A, beta = 97.0 degrees. Crystals of iodinated TmAFP formed elongated hexagons that allowed data to be collected to approximately 1.4 A. These crystals belonged to the space group P6(1) (or P6(5)), with unit-cell dimensions a = 73.85, b = 73.85, c = 53.15 A. There were two molecules per asymmetric unit, which corresponds to V(m) = 2.46 A(3) Da(-1) and 51% solvent content. A twofold non-crystallographic symmetry was evident from self-rotation calculations.