Purification, crystallization and preliminary X-ray analysis of ferredoxin isolated from thermophilic cyanobacterium Mastigocladus laminosus.
Ferredoxins are soluble iron-sulfur proteins that are involved in numerous electron-transfer reactions. Plant-type ferredoxins, which carry a single [2Fe-2S] cluster, serve as the electron acceptors of Photosystem I. The ferredoxin from the thermophilic cyanobacterium Mastigocladus laminosus has unique thermostable properties. The isolated protein is active at temperatures of higher than 338 K. The gene encoding the ferredoxin from M. laminosus was subcloned into an expression vector and overproduced in Escherichia coli. The recombinant protein was purified to near-homogeneity and crystallized using the hanging-drop method. Thin needle-like crystals were grown in several crystallization conditions but were unsuitable for X-ray analysis owing to poor scattering. In order to obtain better diffracting crystals, ferredoxin was purified directly from M. laminosus cells. Single crystals were obtained using 30% PEG 4000, 0.32 M Mg(NO(3))(2), 20 mM Tris-HCl pH 8.2. These crystals diffracted to 1.25 A resolution using synchrotron radiation and were found to belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 28.45, b = 50.93, c = 110.91 A. The asymmetric unit was found to contain two ferredoxin molecules, with a corresponding V(M) of 1.9 A(3) Da(-1) and a solvent content of 34%. The present study indicates that overcoming the poor diffraction of crystals obtained from recombinant protein can be achieved by producing crystals from protein purified from the native host.
Fish
Alexander
A
Department of Plant Science, The Wolfson Centre for Applied Structural Biology, The Hebrew University of Jerusalem, Givat Ram, Israel.
Lebendiker
Mario
M
Nechushtai
Rachel
R
Livnah
Oded
O
D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D000458:Cyanobacteria / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005288:Ferredoxins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D015870:Gene Expression; D005798:Genes, Bacterial; D006358:Hot Temperature; D009682:Magnetic Resonance Spectroscopy
D005288:Ferredoxins