Purification and Crystallization of Chloromuconolactone Dehalogenase ClcF from Rhodococcus opacus 1CP
红球菌属1CP中的氯代粘康酸内酯脱卤素酶CLcF的纯化和结晶化
发布: 2014年04月20日第4卷第8期 DOI: 10.21769/BioProtoc.1107 浏览次数: 10807
评审: Fanglian He
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参见作者原研究论文
The authors used this protocol in:
Apr 2013
Abstract
The protocol describes the generation of variants of chloromuconolactone dehalogenase from Rhodococcus opacus (R. opacus) 1CP. ClcF is a multimeric protein, which catalyses the dechlorination of 5-chloromuconolactone to cis-dienelactone in the 3-chlorocatecholic acid degradation pathway. The protocol describes the workflow for the purification and subsequent crystallization of the enzyme. The used workflow and the described techniques could be easily adapted to any other protein/enzyme intended to be crystallized by the potential user for subsequent structure determination. The protocol does not involve expensive specialized equipment which allows the use in standard laboratories not specially dedicated to macromolecular crystallography.
Keywords: Crystallisation (结晶)
Protein purification (蛋白纯化)
Column chromatography (柱色谱法)
Recombinant protein expression (重组蛋白的表达)
Biochemistry (生物化学)
Materials and Reagents
- Escherichia coli (E. coli) BL21 (DE3)-CP-RIL (Stratagene, catalog number: 230245 )
- E.coli DH5α (Life Technologies, catalog number: 18258012 )
- wtClcF plasmid (not commerically available)
- Forward Primer (not commerically available)
- Reverse Primer (not commerically available)
- dNTP-Mix (20 mM) (Thermo Fisher Scientific, catalog number: AB-0196
- Pfu-Ultra (2.5 U/µl) (Agilent, catalog number: 600385 )
- DpnI (Thermo Fisher Scientific, catalog number: ER1701 )
- LB medium
- Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Carl Roth, catalog number: CN08.1 )
- Tris base (Carl Roth, catalog number: 5429.1 )
- DNase (Sigma-Aldrich, catalog number: DN25-100MG )
- Ethylene glycol
- MgCl2 (Carl Roth, catalog number: 2189.2 )
- PEG 3350 (Sigma-Aldrich, catalog number: 202440-250G )
- Bis-Tris (Carl Roth, catalog number: 9140.1 )
- Liquid nitrogen
- Lysis buffer/Ion exchange (IEX) buffer A/Hydrophobic interaction chromatography (HIC) buffer A (see Recipes)
- HIC High salt buffer (see Recipes)
- Wash buffer (see Recipes)
- Crystallization buffers (see Recipes)
Equipment
- Standard laboratory equipment
- Nanodrop device (Thermo Fisher Scientific)
- CrystalQuickTM 96 well crystallization plate Greiner 609101(Jena Bioscience, catalog number: CPL-118S )
- Linbro crystallization plate (Jena Bioscience, catalog number: CPL-101S )
- 22 mm circular cover slides (siliconized) (Jena Bioscience, catalog number: CSL-106 )
- Bayer silicone grease (Jena Bioscience, catalog number: CGR-101 )
- Äkta purification system (GE Healthcare)
- HiTrap Q sepharose column (1 ml) (GE Healthcare, catalog number: 17-5053-01 )
- HiTrap phenyl-sepharose (1 ml) (GE Healthcare, catalog number: 17-1351-01 )
- Stereo microscope (Leica Microsystems, model: MZ12 )
- Eppendorf tubes
- Pipetting robot (if available)
- 37 °C incubator
- Centrifuge
- Vivaspin concentrator (MWCO 30 kDa) (EMD Millipore)
Procedure
文章信息
版权信息
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Roth, C., Gröning, J. A. D., Kaschabek, S. R., Schlömann, M. and Sträter, N. (2014). Purification and Crystallization of Chloromuconolactone Dehalogenase ClcF from Rhodococcus opacus 1CP. Bio-protocol 4(8): e1107. DOI: 10.21769/BioProtoc.1107.
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分类
微生物学 > 微生物生物化学 > 蛋白质
生物化学 > 蛋白质 > 结构
生物化学 > 蛋白质 > 分离和纯化
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