Open AccessCloning, expression, purification, crystallization and preliminary X‐ray characterization of the full‐length single‐stranded DNA‐binding protein from the hyperthermophilic bacterium Aquifex aeolicus
Author(s) -
Clarke David J.,
Northey Christopher G.,
Mack Lynsey A.,
McNae Iain W.,
Alexeev Dmitriy,
Sawyer Lindsay,
Campopiano Dominic J.
Publication year - 2004
Publication title -
acta crystallographica section d
Language(s) - English
Resource type - Journals
ISSN - 1399-0047
DOI - 10.1107/s0907444904020530
Subject(s) - aquifex aeolicus , hyperthermophile , tetramer , crystallography , dna , escherichia coli , chemistry , dna replication , biochemistry , archaea , gene , enzyme
Single‐stranded DNA‐binding (SSB) proteins stabilize single‐stranded DNA, which is exposed by separation of the duplex during DNA replication, recombination and repair. The SSB protein from the hyperthermophile Aquifex aeolicus has been overexpressed in Escherichia coli , purified and characterized and crystals of the full‐length protein (147 amino acids; M r 17 131.20) have been grown by vapour diffusion from ammonium sulfate pH 7.5 in both the absence and presence of ssDNA [dT(pT) 68 ]. All crystals diffract to around 2.9 Å resolution and those without bound DNA (native) belong to space group P 2 1 , with two tetramers in the asymmetric unit and unit‐cell parameters a = 80.97, b = 73.40, c = 109.76 Å, β = 95.11°. Crystals containing DNA have unit‐cell parameters a = 108.65, b = 108.51, c = 113.24 Å and could belong to three closely related space groups ( I 222, I 2 1 2 1 2 1 or I 4 1 ) with one tetramer in the asymmetric unit. Electrospray mass spectrometry of the crystals confirmed that the protein was intact. Molecular replacement with a truncated E. coli SSB structure has revealed the position of the molecules in the unit cell and refinement of both native and DNA‐bound forms is under way.