Expression, purification and crystallization of 2-oxo-hept-4-ene-1,7-dioate hydratase (HpcG) from Escherichia coli C
Journal Article
·
· Acta Crystallographica. Section F
- Department of Biological Sciences, University of Warwick, Coventry CV4 7AL (United Kingdom)
- Division of Protein Design, Yokohama City University, Suehiro 1-7-29, Yokohama, Kanagawa 230-0045 (Japan)
The gene encoding HpcG from the homoprotocatechuate (4-hydroxyphenylacetic acid) degradative pathway of E. coli C has been cloned and expressed and the protein has been purified. Crystals obtained from the purified recombinant enzyme, belonging to a tetragonal space group, diffracted to a resolution of 2.1 Å. The gene encoding 2-oxo-hept-3-ene-1,7-dioic acid (OHED) hydratase (HpcG) was cloned into the high-expression plasmid pET26b and overexpressed in Escherichia coli BL21(DE3). The enzyme was purified in three steps to greater than 95% purity prior to crystallization. Crystals were obtained by the hanging-drop vapour-diffusion method at 277 K in a number of screening conditions. Crystals measuring up to 1.5 mm in their longest dimension were grown from solutions containing polyethylene glycol 20 000. The crystals belonged to space group P4{sub 1}2{sub 1}2 or P4{sub 3}2{sub 1}2, with unit-cell parameters a = 136, b = 136, c = 192 Å. A complete data set was collected to 2.1 Å from a single cryocooled crystal at 100 K using synchrotron radiation.
- OSTI ID:
- 22356355
- Journal Information:
- Acta Crystallographica. Section F, Journal Name: Acta Crystallographica. Section F Journal Issue: Pt 10 Vol. 62; ISSN ACSFCL; ISSN 1744-3091
- Country of Publication:
- United Kingdom
- Language:
- English
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