Cloning, expression, purification and characterization of a DsbA-like protein from Wolbachia pipientis.
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, alpha-DsbA1 and alpha-DsbA2, that might catalyze these steps. In this work we focussed on the 234 residue protein alpha-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 alpha-DsbA1 for both dithiol oxidants (E. coli DsbA, 12%) and disulfide isomerases (E. coli DsbC, 14%) is similar. We therefore sought to establish whether alpha-DsbA1 is an oxidant or an isomerase based on functional activity. The purified alpha-DsbA1 was active in an oxidoreductase assay but had little isomerase activity, indicating that alpha-DsbA1 is DsbA-like rather than DsbC-like. This work represents the first successful example of the characterization of a recombinant Wolbachia protein. Purified alpha-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.
Kurz
Mareike
M
Institute for Molecular Bioscience, The University of Queensland, Brisbane, Qld 4072, Australia.
Iturbe-Ormaetxe
Iñaki
I
Jarrott
Russell
R
Cowieson
Nathan
N
Robin
Gautier
G
Jones
Alun
A
King
Gordon J
GJ
Frei
Patrick
P
Glockshuber
Rudi
R
O'Neill
Scott L
SL
Heras
Begoña
B
Martin
Jennifer L
JL
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D019704:Protein Disulfide-Isomerases / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D016415:Sequence Alignment; D020577:Wolbachia / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D019704:Protein Disulfide-Isomerases