Cloning, expression, and purification of Brucella suis outer membrane proteins

Ding, XZ; Bhattacharjee, A; Nikolich, MP; Paulsen, IT; Myers, G; Seshadri, R; Hoover, DL

Cloning, expression, and purification of Brucella suis outer membrane proteins

Ding, XZ Bhattacharjee, A Nikolich, MP Paulsen, IT Myers, G

Seshadri, R Hoover, DL

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Publication Type:: Journal Article
Citation:: Protein Expression and Purification, 2005, 40 (1), pp. 134 - 141
Issue Date:: 2005-01-01

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Field	Value	Language
dc.contributor.author	Ding, XZ	en_US
dc.contributor.author	Bhattacharjee, A	en_US
dc.contributor.author	Nikolich, MP	en_US
dc.contributor.author	Paulsen, IT	en_US
dc.contributor.author	Myers, G https://orcid.org/0000-0002-4756-4810	en_US
dc.contributor.author	Seshadri, R	en_US
dc.contributor.author	Hoover, DL	en_US
dc.date.issued	2005-01-01	en_US
dc.identifier.citation	Protein Expression and Purification, 2005, 40 (1), pp. 134 - 141	en_US
dc.identifier.issn	1046-5928	en_US
dc.identifier.uri	http://hdl.handle.net/10453/39761
dc.description.abstract	Brucella, an aerobic, nonsporeforming, nonmotile Gram-negative coccobacillus, is a NIH/CDC category B bioterror threat agent that causes incapacitating human illness. Medical defense against the bioterror threat posed by Brucella would be strengthened by development of a human vaccine and improved diagnostic tests. Central to advancement of these goals is discovery of bacterial constituents that are immunogenic or antigenic for humans. Outer membrane proteins (OMPs) are particularly attractive for this purpose. In this study, we cloned, expressed, and purified seven predicted OMPs of Brucella suis. The recombinant proteins were fused with 6-His and V5 epitope tags at their C termini to facilitate detection and purification. The B. suis surface genes were PCR synthesized based on their ORF sequences and directly cloned into an entry vector. The recombinant entry constructs were propagated in TOP 10 cells, recombined into a destination vector, pET-DEST42, then transformed into Escherichia coli BL21 cells for IPTG-induced protein expression. The expressed recombinant proteins were confirmed with Western blot analysis using anti-6-His antibody conjugated with alkaline phosphatase. These B. suis OMPs were captured and purified using a HisGrab plate. The purified recombinant proteins were examined for their binding activity with antiserum. Serum derived from a rabbit immunized intramuscularly with dialyzed cell lysate of Brucella rough mutant WRR51. The OMPs were screened using the rabbit antiserum and purified IgG. The results suggested that recombinant B. suis OMPs were successfully cloned, expressed and purified. Some of the expressed OMPs showed high binding activity with immunized rabbit antiserum. © 2005 Elsevier Inc. All rights reserved.	en_US
dc.relation.ispartof	Protein Expression and Purification	en_US
dc.relation.isbasedon	10.1016/j.pep.2004.12.017	en_US
dc.subject.classification	Biochemistry & Molecular Biology	en_US
dc.subject.mesh	Animals	en_US
dc.subject.mesh	Humans	en_US
dc.subject.mesh	Brucella suis	en_US
dc.subject.mesh	Escherichia coli	en_US
dc.subject.mesh	Bacterial Outer Membrane Proteins	en_US
dc.subject.mesh	Cloning, Molecular	en_US
dc.subject.mesh	Base Sequence	en_US
dc.subject.mesh	Molecular Sequence Data	en_US
dc.title	Cloning, expression, and purification of Brucella suis outer membrane proteins	en_US
dc.type	Journal Article
utslib.citation.volume	1	en_US
utslib.citation.volume	40	en_US
utslib.for	0601 Biochemistry and Cell Biology	en_US
utslib.for	0699 Other Biological Sciences	en_US
pubs.embargo.period	Not known	en_US
pubs.organisational-group	/University of Technology Sydney
pubs.organisational-group	/University of Technology Sydney/Faculty of Science
pubs.organisational-group	/University of Technology Sydney/Strength - ithree - Institute of Infection, Immunity and Innovation
utslib.copyright.status	closed_access
pubs.issue	1	en_US
pubs.publication-status	Published	en_US
pubs.volume	40	en_US

Abstract:

Brucella, an aerobic, nonsporeforming, nonmotile Gram-negative coccobacillus, is a NIH/CDC category B bioterror threat agent that causes incapacitating human illness. Medical defense against the bioterror threat posed by Brucella would be strengthened by development of a human vaccine and improved diagnostic tests. Central to advancement of these goals is discovery of bacterial constituents that are immunogenic or antigenic for humans. Outer membrane proteins (OMPs) are particularly attractive for this purpose. In this study, we cloned, expressed, and purified seven predicted OMPs of Brucella suis. The recombinant proteins were fused with 6-His and V5 epitope tags at their C termini to facilitate detection and purification. The B. suis surface genes were PCR synthesized based on their ORF sequences and directly cloned into an entry vector. The recombinant entry constructs were propagated in TOP 10 cells, recombined into a destination vector, pET-DEST42, then transformed into Escherichia coli BL21 cells for IPTG-induced protein expression. The expressed recombinant proteins were confirmed with Western blot analysis using anti-6-His antibody conjugated with alkaline phosphatase. These B. suis OMPs were captured and purified using a HisGrab plate. The purified recombinant proteins were examined for their binding activity with antiserum. Serum derived from a rabbit immunized intramuscularly with dialyzed cell lysate of Brucella rough mutant WRR51. The OMPs were screened using the rabbit antiserum and purified IgG. The results suggested that recombinant B. suis OMPs were successfully cloned, expressed and purified. Some of the expressed OMPs showed high binding activity with immunized rabbit antiserum. © 2005 Elsevier Inc. All rights reserved.

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http://hdl.handle.net/10453/39761