Open AccessCloning, overexpression, purification, crystallization and preliminary X‐ray analysis of 3‐ketosteroid Δ 4 ‐(5α)‐dehydrogenase from Rhodococcus jostii RHA1
Author(s) -
van Oosterwijk Niels,
Knol Jan,
Dijkhuizen Lubbert,
van der Geize Robert,
Dijkstra Bauke W.
Publication year - 2011
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309111028727
Subject(s) - ketosteroid , rhodococcus , escherichia coli , dehydrogenase , chemistry , affinity chromatography , cloning (programming) , enzyme , biology , biochemistry , gene , isomerase , computer science , programming language
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 4 ‐(5α)‐dehydrogenase [Δ 4 ‐(5α)‐KSTD] from R. jostii RHA1 was cloned and overexpressed in Escherichia coli . His‐tagged Δ 4 ‐(5α)‐KSTD enzyme was purified by Ni 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 C 222 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.