title: Fed batch fermentation and purification strategy for high yield production of Brucella melitensis recombinant Omp 28 kDa protein and its application in disease diagnosis.
abstract: Brucellosis is a disease caused by bacteria belonging to the genus Brucella. It affects cattle, goat, sheep, dog and humans. The serodiagnosis of brucellosis involves detection of antibodies generated against the LPS or whole cell bacterial extracts, however these tests lack sensitivity and specificity. The present study was performed to optimize the culture condition for the production of recombinant Brucella melitensis outer membrane protein 28 kDa protein in E.coli via fed batch fermentation. Expression was induced with 1.5mM isopropyl β thiogalactoside and the expressed recombinant protein was purified using Ni-NTA affinity chromatography. After fed-batch fermentation the dry cell weight of 17.81 g/L and a purified protein yield of 210.10 mg/L was obtained. The purified Brucella melitensis recombinant Omp 28 kDa protein was analyzed through SDS- poly acrylamide gel electrophoresis and western blotting. The obtained recombinant protein was evaluated for its diagnostic application through Indirect ELISA using brucellosis suspected human sera samples. Our results clearly indicate that recombinant Omp28 produced via fed batch fermentation has immense potential as a diagnostic reagent that could be employed in sero monitoring of brucellosis.
authors.affiliation: Bioprocess scale-up facility, Defence Research and Development Estt. Jhansi Road, Gwalior, 474002, India.
authors.forename: B S
authors.initials: BS
authors.lastname: Karothia
authors.forename: T N
authors.initials: TN
authors.lastname: Athmaram
authors.forename: Thavaselvam
authors.initials: T
authors.lastname: D
authors.forename: Kumar
authors.initials: K
authors.lastname: Ashu
authors.forename: Sapna
authors.initials: S
authors.lastname: Tiwari
authors.forename: Anil K
authors.initials: AK
authors.lastname: Singh
authors.forename: K
authors.initials: K
authors.lastname: Sathyaseelan
authors.forename: N
authors.initials: N
authors.lastname: Gopalan
mesh_terms: D000907:Antibodies, Bacterial / Q000097:blood / Q000276:immunology / Q000302:isolation & purification; D019149:Bioreactors / Q000382:microbiology; D015153:Blotting, Western; D017347:Brucella melitensis / Q000235:genetics* / Q000378:metabolism; D002006:Brucellosis / Q000175:diagnosis; D003470:Culture Media; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005285:Fermentation; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D000907:Antibodies, Bacterial; C106040:BP26 protein, Brucella; D003470:Culture Media; D008565:Membrane Proteins; D011994:Recombinant Proteins