Cloning, expression, and purification of Brucella suis outer membrane proteins.
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.
Ding
Xuan Z
XZ
Division of CD&I, Department of Bacterial Diseases, Walter Reed Army Institute of Research, Washington, DC, USA. tom.ding@na.amedd.army.mil
Bhattacharjee
Apurba
A
Nikolich
Mikeljon P
MP
Paulsen
Ian T
IT
Myers
Gary
G
Seshadri
Rekha
R
L Hoover
David
D
D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D001483:Base Sequence; D041021:Brucella suis / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data
D001425:Bacterial Outer Membrane Proteins