Cloning, expression, purification, crystallization and preliminary X-ray characterization of the full-length single-stranded DNA-binding protein from the hyperthermophilic bacterium Aquifex aeolicus

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 a...

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Bibliographic Details
Published in:	Acta crystallographica. Section D, Biological crystallography. Vol. 60; no. 11; pp. 2009 - 2012
Main Authors:	Clarke, David J., Northey, Christopher G., Mack, Lynsey A., McNae, Iain W., Alexeev, Dmitriy, Sawyer, Lindsay, Campopiano, Dominic J.
Format:	Journal Article
Language:	English
Published:	5 Abbey Square, Chester, Cheshire CH1 2HU, England Munksgaard International Publishers 01-11-2004
Subjects:	Amino Acid Sequence Aquifex aeolicus Bacteria - chemistry Bacterial Proteins - chemistry Bacterial Proteins - genetics Bacterial Proteins - isolation & purification Bacterial Proteins - metabolism Cloning, Molecular Crystallization Crystallography, X-Ray DNA replication DNA, Single-Stranded - metabolism DNA-Binding Proteins - chemistry DNA-Binding Proteins - genetics DNA-Binding Proteins - isolation & purification DNA-Binding Proteins - metabolism Gene Expression Hot Temperature hyperthermophile Molecular Sequence Data Sequence Alignment single-stranded DNA-binding protein Spectrometry, Mass, Electrospray Ionization
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Summary:	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; Mr 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 P21, 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 (I222, I212121 or I41) 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.
Bibliography:	ArticleID:AYDBW5042 ark:/67375/WNG-S8CD0JSJ-7 istex:7DA61BA36BE077890F547633F1A526BA31CB6A7D ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	1399-0047 0907-4449 1399-0047
DOI:	10.1107/S0907444904020530