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Cloning, overexpression, purification, crystallization and preliminary X-ray analysis of 3-ketosteroid Δ{sup 4}-(5α)-dehydrogenase from Rhodococcus jostii RHA1

Abstract

The gene for 3-ketosteroid Δ{sup 4}-(5α)-dehydrogenase from R. jostii RHA1 was cloned and overexpressed in E. coli and the protein product was purified and crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to space group C222{sub 1} and diffraction data were collected to a resolution of 1.6 Å. 3-Ketosteroid dehydrogenases are flavoproteins which play key roles in steroid ring degradation. The enzymes are abundantly present in actinobacteria, including the catabolic powerhouse Rhodococcus jostii and the pathogenic species R. equi and Mycobacterium tuberculosis. The gene for 3-ketosteroid Δ{sup 4}-(5α)-dehydrogenase [Δ{sup 4}-(5α)-KSTD] from R. jostii RHA1 was cloned and overexpressed in Escherichia coli. His-tagged Δ{sup 4}-(5α)-KSTD enzyme was purified by Ni{sup 2+}–NTA affinity chromatography, anion-exchange chromatography and size-exclusion chromatography and was crystallized using the hanging-drop vapour-diffusion method. Seeding greatly improved the number of crystals obtained. The crystals belonged to space group C222{sub 1}, with unit-cell parameters a = 99.2, b = 114.3, c = 110.2 Å. Data were collected to a resolution of 1.6 Å.
Authors:
Oosterwijk, Niels van; Knol, Jan; Dijkhuizen, Lubbert; Geize, Robert van der; Dijkstra, Bauke W., E-mail: b.w.dijkstra@rug.nl [1] 
  1. University of Groningen, Nijenborgh 7, 9747 AG Groningen (Netherlands)
Publication Date:
Oct 01, 2011
Product Type:
Journal Article
Resource Relation:
Journal Name: Acta Crystallographica. Section F; Journal Volume: 67; Journal Issue: Pt 10; Other Information: PMCID: PMC3212380; PMID: 22102045; PUBLISHER-ID: uo5029; OAI: oai:pubmedcentral.nih.gov:3212380; Copyright (c) International Union of Crystallography 2011; Country of input: International Atomic Energy Agency (IAEA)
Subject:
75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND SUPERFLUIDITY; AFFINITY; CRYSTALLIZATION; CRYSTALS; DIFFRACTION; DIFFUSION; ESCHERICHIA COLI; RESOLUTION; RINGS; SPACE GROUPS
OSTI ID:
22371576
Country of Origin:
United Kingdom
Language:
English
Other Identifying Numbers:
Journal ID: ISSN 1744-3091; CODEN: ACSFCL; Other: PII: S1744309111028727; TRN: GB15$1377076944
Availability:
Available from http://dx.doi.org/10.1107/S1744309111028727; Available from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3212380
Submitting Site:
INIS
Size:
page(s) 1269-1273
Announcement Date:
Aug 06, 2015

Citation Formats

Oosterwijk, Niels van, Knol, Jan, Dijkhuizen, Lubbert, Geize, Robert van der, and Dijkstra, Bauke W., E-mail: b.w.dijkstra@rug.nl. Cloning, overexpression, purification, crystallization and preliminary X-ray analysis of 3-ketosteroid Δ{sup 4}-(5α)-dehydrogenase from Rhodococcus jostii RHA1. United Kingdom: N. p., 2011. Web. doi:10.1107/S1744309111028727.
Oosterwijk, Niels van, Knol, Jan, Dijkhuizen, Lubbert, Geize, Robert van der, & Dijkstra, Bauke W., E-mail: b.w.dijkstra@rug.nl. Cloning, overexpression, purification, crystallization and preliminary X-ray analysis of 3-ketosteroid Δ{sup 4}-(5α)-dehydrogenase from Rhodococcus jostii RHA1. United Kingdom. doi:10.1107/S1744309111028727.
Oosterwijk, Niels van, Knol, Jan, Dijkhuizen, Lubbert, Geize, Robert van der, and Dijkstra, Bauke W., E-mail: b.w.dijkstra@rug.nl. 2011. "Cloning, overexpression, purification, crystallization and preliminary X-ray analysis of 3-ketosteroid Δ{sup 4}-(5α)-dehydrogenase from Rhodococcus jostii RHA1." United Kingdom. doi:10.1107/S1744309111028727. https://www.osti.gov/servlets/purl/10.1107/S1744309111028727.
@misc{etde_22371576,
title = {Cloning, overexpression, purification, crystallization and preliminary X-ray analysis of 3-ketosteroid Δ{sup 4}-(5α)-dehydrogenase from Rhodococcus jostii RHA1}
author = {Oosterwijk, Niels van, Knol, Jan, Dijkhuizen, Lubbert, Geize, Robert van der, and Dijkstra, Bauke W., E-mail: b.w.dijkstra@rug.nl}
abstractNote = {The gene for 3-ketosteroid Δ{sup 4}-(5α)-dehydrogenase from R. jostii RHA1 was cloned and overexpressed in E. coli and the protein product was purified and crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to space group C222{sub 1} and diffraction data were collected to a resolution of 1.6 Å. 3-Ketosteroid dehydrogenases are flavoproteins which play key roles in steroid ring degradation. The enzymes are abundantly present in actinobacteria, including the catabolic powerhouse Rhodococcus jostii and the pathogenic species R. equi and Mycobacterium tuberculosis. The gene for 3-ketosteroid Δ{sup 4}-(5α)-dehydrogenase [Δ{sup 4}-(5α)-KSTD] from R. jostii RHA1 was cloned and overexpressed in Escherichia coli. His-tagged Δ{sup 4}-(5α)-KSTD enzyme was purified by Ni{sup 2+}–NTA affinity chromatography, anion-exchange chromatography and size-exclusion chromatography and was crystallized using the hanging-drop vapour-diffusion method. Seeding greatly improved the number of crystals obtained. The crystals belonged to space group C222{sub 1}, with unit-cell parameters a = 99.2, b = 114.3, c = 110.2 Å. Data were collected to a resolution of 1.6 Å.}
doi = {10.1107/S1744309111028727}
journal = {Acta Crystallographica. Section F}
issue = {Pt 10}
volume = {67}
journal type = {AC}
place = {United Kingdom}
year = {2011}
month = {Oct}
}