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. 2007 Jun 1;63(Pt 6):512-5.
doi: 10.1107/S1744309107022154. Epub 2007 May 12.

Crystallization and preliminary crystallographic analysis of cysteine synthase from Entamoeba histolytica

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Crystallization and preliminary crystallographic analysis of cysteine synthase from Entamoeba histolytica

Chinthalapudi Krishna et al. Acta Crystallogr Sect F Struct Biol Cryst Commun. .

Abstract

Entamoeba histolytica, the causative agent of human amoebiasis, is essentially anaerobic, requiring a small amount of oxygen for growth. It cannot tolerate the higher concentration of oxygen present in human tissues or blood. However, during tissue invasion it is exposed to a higher level of oxygen, leading to oxygen stress. Cysteine, which is a vital thiol in E. histolytica, plays an essential role in its oxygen-defence mechanisms. The major route of cysteine biosynthesis in this parasite is the condensation of O-acetylserine with sulfide by the de novo cysteine-biosynthetic pathway, which involves cysteine synthase (EhCS) as a key enzyme. In this study, EhCS was cloned, expressed in Escherichia coli and purified by affinity and size-exclusion chromatography. The purified protein was crystallized in space group P4(1) with two molecules per asymmetric unit and a complete data set was collected to a resolution of 1. 86 A. A molecular-replacement solution was obtained using the Salmonella typhimurium O-acetylserine sulfhydrylase structure as a probe and had a correlation coefficient of 37. 7% and an R factor of 48. 8%. VSports手机版.

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Figure 1
Figure 1
SDS–PAGE showing fractions purified by Ni–NTA and gel-filtration chromatography. Proteins were separated on 12% SDS–PAGE and stained with Coomassie Brilliant Blue. Lane 1, molecular-weight markers (kDa); lanes 2–4, Ni–NTA purified fractions; lanes 5–6, gel-filtration purified fractions.
Figure 2
Figure 2
(a) Single elongated hexagonal shaped crystals of EhCS obtained along with protein precipitate at 289 K using the hanging-drop vapour-diffusion method. (b) Diffraction pattern of EhCS crystals to 1.86 Å resolution. The data were collected using a Rigaku MicroMax-007 generator and a MAR imaging plate. The imaging plate was adjusted to a distance of 150 mm and the crystals were exposed for 90 s per frame. Diffraction spots were observed to the edges of the image plate, as shown in the enlargement.

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