Cloning, expression, purification and characterization of a DsbA-like protein from Wolbachia pipientis

Kurz, Mareike, Iturbe-Ormaetxe, Inaki, Jarrott, Russell, Cowieson, Nathan, Robin, Gautier, Jones, Alun, King, Gordon J., Frei, Patrick, Glockshuber, Rudi, O'Neill, Scott L., Heras, Begona and Martin, Jennifer L. (2008) Cloning, expression, purification and characterization of a DsbA-like protein from Wolbachia pipientis. Protein Expression and Purification, 59 2: 266-273. doi:10.1016/j.pep.2008.02.008

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Author Kurz, Mareike
Iturbe-Ormaetxe, Inaki
Jarrott, Russell
Cowieson, Nathan
Robin, Gautier
Jones, Alun
King, Gordon J.
Frei, Patrick
Glockshuber, Rudi
O'Neill, Scott L.
Heras, Begona
Martin, Jennifer L.
Title Cloning, expression, purification and characterization of a DsbA-like protein from Wolbachia pipientis
Formatted title
Cloning, expression, purification and characterization of a DsbA-like protein from Wolbachia pipientis
Journal name Protein Expression and Purification   Check publisher's open access policy
ISSN 1046-5928
1096-0279
Publication date 2008-06
Sub-type Article (original research)
DOI 10.1016/j.pep.2008.02.008
Open Access Status File (Author Post-print)
Volume 59
Issue 2
Start page 266
End page 273
Total pages 8
Editor R. R. Burgess
Place of publication San Diego, USA
Publisher Academic Press
Collection year 2009
Language eng
Subject C1
960499 Control of Pests, Diseases and Exotic Species not elsewhere classified
060112 Structural Biology (incl. Macromolecular Modelling)
Abstract Wolbachia pipientis are obligate endosymbionts that infect a wide range of insect and other arthropod species. They act as reproductive parasites by manipulating the host reproduction machinery to enhance their own transmission. This unusual phenotype is thought to be a consequence of the actions of secreted Wolbachia proteins that are likely to contain disulfide bonds to stabilize the protein structure. In bacteria, the introduction or isomerization of disulfide bonds in proteins is catalyzed by Dsb proteins. The Wolbachia genome encodes two proteins, a-DsbA1 and a-DsbA2, that might catalyze these steps. In this work we focussed on the 234 residue protein a-DsbA1; the gene was cloned and expressed in Escherichia coli, the protein was purified and its identity confirmed by mass spectrometry. The sequence identity of a-DsbA1 for both dithiol oxidants(E. coli DsbA, 12%) and disulfide isomerases(E. coli DsbC, 14%) is similar. We therefore sought to establish whether a-DsbA1 is an oxidant or an isomerase based on functional activity. The purified a-DsbA1 was active in an oxidoreductase assay but had little isomerase activity, indicating that a-DsbA1 is DsbA-like rather than DsbC-like. This work represents the first successful example of the characterization of a recombinant Wolbachia protein. Purified a-DsbA1 will now be used in further functional studies to identify protein substrates that could help explain the molecular basis for the unusual Wolbachia phenotypes, and in structural studies to explore its relationship to other disulfide oxidoreductase proteins. Copyright © 2008 Elsevier Inc
Keyword Oxidoreductase
a-Proteobacteria
Protein disulfide oxidant
Q-Index Code C1
Q-Index Status Confirmed Code

 
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Created: Mon, 09 Feb 2009, 18:48:48 EST by Gail Walter on behalf of Institute for Molecular Bioscience