Expression, Purification and Crystallization of Thermostable Mutant of Cutinase Est1 from Thermobifida alba

Kitadokoro, Kengo and Matsui, Shingo and Osokoshi, Ryouhei and Nakata, Kensuke and Kamitani, Shigeki (2018) Expression, Purification and Crystallization of Thermostable Mutant of Cutinase Est1 from Thermobifida alba. Advances in Bioscience and Biotechnology, 09 (05). pp. 215-223. ISSN 2156-8456

[thumbnail of ABB_2018052915395598.pdf] Text
ABB_2018052915395598.pdf - Published Version

Download (711kB)

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.

Item Type: Article
Subjects: Journal Eprints > Biological Science
Depositing User: Managing Editor
Date Deposited: 21 Jan 2023 05:33
Last Modified: 10 Jul 2024 14:00
URI: http://repository.journal4submission.com/id/eprint/972

Actions (login required)

View Item
View Item