Expression, Purification and Crystallization of Thermostable Mutant of Cutinase Est1 from <i>Thermobifida alba</i>

A double mutantEst1, which is a plastic degrading cutinase-type esterase in Thermobifida alba , has been over-expressed in Escherichia coli . The recombinant protein was purified by a two-step protocol involving immobilized metal affinity chromatography and cation-exchange chromatography, yielding 120 mg of protein per liter of bacterial culture. Crystals have been obtained by using the sitting-drop vapor-diffusion technique. Native diffraction data to 1.37 A resolution were obtained at the BL44XU beam line of SPring-8 from a flash-frozen crystal at 100 K. The crystals belong to space group C 2, with unit-cell parameters a = 127.2 A, b = 42.1 A, c = 63.2 A, β = 114.7°, likely containing one Est1 double mutant (296 residues) per asymmetric unit.