title: Optimization and efficient purification of recombinant Omp28 protein of Brucella melitensis using Triton X-100 and β-mercaptoethanol.
abstract: The high level expression of recombinant proteins in Escherichia coli often leads to the formation of inclusion bodies that contain most of the expressed protein held together by non-covalent forces. The inclusion bodies are usually solubilized using strong denaturing agents like urea and guanidium hydrochloride. In this study recombinant Omp28 (rOmp28) protein of Brucella melitensis was expressed in two different vector systems and further efficient purification of the protein was done by modification in buffers to improve the yield and purity. Different concentrations of Triton X-100 and β-mercaptoethanol were optimized for the solubilization of inclusion bodies. The lysis buffer with 8M urea alone was not sufficient to solubilize the inclusion bodies. It was found that the use of 1% Triton X-100 and 20mM β-mercaptoethanol in lysis and wash buffers used at different purification steps under denaturing conditions increased the yield of purified rOmp28 protein. The final yield of purified protein obtained with modified purification protocol under denaturing conditions was 151 and 90mg/l of the culture or 11.8 and 9.37mg/g of wet weight of cells in pQE30UA and pET28a(+) vector respectively. Thus modified purification protocol yielded more than threefold increase of protein in pQE30UA as compared with purification by conventional methods.
authors.affiliation: Microbiology Division, Defence Research and Development Establishment, Jhansi Road, Gwalior 474002, India.
authors.forename: Ashu
authors.initials: A
authors.lastname: Kumar
authors.forename: Sapana
authors.initials: S
authors.lastname: Tiwari
authors.forename: Duraipandian
authors.initials: D
authors.lastname: Thavaselvam
authors.forename: Kannusamy
authors.initials: K
authors.lastname: Sathyaseelan
authors.forename: Archana
authors.initials: A
authors.lastname: Prakash
authors.forename: Anita
authors.initials: A
authors.lastname: Barua
authors.forename: Sonia
authors.initials: S
authors.lastname: Arora
authors.forename: M
authors.initials: M
authors.lastname: Kameswara Rao
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D017347:Brucella melitensis / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005822:Genetic Vectors; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008623:Mercaptoethanol / Q000737:chemistry*; D017830:Octoxynol / Q000737:chemistry*; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: C106040:BP26 protein, Brucella; D001426:Bacterial Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; D008623:Mercaptoethanol; D017830:Octoxynol