Expression, purification, crystallization and initial X-ray diffraction analysis of thiol peroxidase from Yersinia
Mads Gabrielsen1, Caroline E Zetterström, Dai Wang
1Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, Glasgow Biomedical Research Centre, Glasgow G12 8QQ, Scotland. mads.gabrielsen@glasgow.ac.uk
Summary
Thiol peroxidase, an atypical 2-Cys peroxiredoxin, was expressed and purified. Crystallization trials successfully produced three distinct crystal forms, aiding further structural studies of this enzyme.
Area of Science:
- Biochemistry
- Structural Biology
- Enzymology
Background:
- Thiol peroxidase is an atypical 2-Cys peroxiredoxin.
- This enzyme plays a role in reducing alkyl hydroperoxides.
Purpose of the Study:
- To recombinantly express and purify wild-type and C61S mutant thiol peroxidase.
- To investigate the crystallization properties of thiol peroxidase.
Main Methods:
- Recombinant protein expression in Escherichia coli.
- Purification using nickel-affinity chromatography.
- Crystallization trials under various conditions.
Main Results:
- Wild-type and C61S mutant thiol peroxidase were successfully expressed and purified.
- Three distinct crystal forms were obtained in different space groups (P2(1), P6(4), and P2(1)2(1)2(1)).
- Crystallization was achieved both with and without DTT.
Conclusions:
- The successful purification and crystallization of thiol peroxidase provide a foundation for future structural and functional analyses.
- The obtained crystal forms offer opportunities to elucidate the enzyme's mechanism of action.
