TITLE:
Expression, Purification and Crystallization of Thermostable Mutant of Cutinase Est1 from Thermobifida alba
AUTHORS:
Kengo Kitadokoro, Shingo Matsui, Ryouhei Osokoshi, Kensuke Nakata, Shigeki Kamitani
KEYWORDS:
Cutinase, Themobifida alba, Plastic Degrading Esterase, Crystallization, High Resolution Crystallographic Data
JOURNAL NAME:
Advances in Bioscience and Biotechnology,
Vol.9 No.5,
May
30,
2018
ABSTRACT: 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 Å
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 C2, with
unit-cell parameters a = 127.2 Å, b = 42.1 Å, c = 63.2 Å, β = 114.7°, likely containing one Est1 double mutant (296
residues) per asymmetric unit.