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Expression, Purification and Crystallization of Thermostable Mutant of Cutinase Est1 from <i>Thermobifida alba</i>
Departmentof Biomolecular Engineering, Graduate School of Science and Technology, Kyoto Institute of Technology, Kyoto, Japan
Departmentof Biomolecular Engineering, Graduate School of Science and Technology, Kyoto Institute of Technology, Kyoto, Japan
Departmentof Biomolecular Engineering, Graduate School of Science and Technology, Kyoto Institute of Technology, Kyoto, Japan
Departmentof Biomolecular Engineering, Graduate School of Science and Technology, Kyoto Institute of Technology, Kyoto, Japan
Graduate School of Comprehensive Rehabilitation, College of Health and Human Sciences, Osaka Prefecture University, Osaka, Japan
- 1 Departmentof Biomolecular Engineering, Graduate School of Science and Technology, Kyoto Institute of Technology, Kyoto, Japan
- 2 Departmentof Biomolecular Engineering, Graduate School of Science and Technology, Kyoto Institute of Technology, Kyoto, Japan
- 3 Departmentof Biomolecular Engineering, Graduate School of Science and Technology, Kyoto Institute of Technology, Kyoto, Japan
- 4 Departmentof Biomolecular Engineering, Graduate School of Science and Technology, Kyoto Institute of Technology, Kyoto, Japan
- 5 Graduate School of Comprehensive Rehabilitation, College of Health and Human Sciences, Osaka Prefecture University, Osaka, Japan
Advances in Bioscience and Biotechnology·Volume 09 (2018)·Pages 215–223·Published 9 May 2018·DOI10.4236/abb.2018.95015
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Abstract
A double mutantEst1, which is a plastic degrading cutinase-type esterase in <i> Thermobifida alba </i> , has been over - expressed in <i> Escherichia coli </i> . 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 <i> C </i> 2, with unit-cell parameters <i> a </i> = 127.2 Å , <i> b </i> = 42.1 Å , <i> c </i> = 63.2 Å , <i> β </i> = 114.7 ° , likely containing one Est1 double mutant (296 residues) per asymmetric unit.
KeywordsCutinase<i>Themobifida alba</i>Plastic Degrading EsteraseCrystallizationHigh Resolution Crystallographic Data
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