Optimization and efficient purification of recombinant Omp28 protein of Brucella melitensis using Triton X-100 and β-mercaptoethanol

• Published in: Protein expression and purification 2012-06, Vol.83 (2), p.226-232
• Main Authors: Kumar, Ashu, Tiwari, Sapana, Thavaselvam, Duraipandian, Sathyaseelan, Kannusamy, Prakash, Archana, Barua, Anita, Arora, Sonia, Kameswara Rao, M.
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; Brucella melitensis; Brucella melitensis - genetics; Brucella melitensis - metabolism; Brucellosis; Cloning, Molecular; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Genetic Vectors; Membrane Proteins - chemistry; Membrane Proteins - genetics; Membrane Proteins - isolation & purification; Membrane Proteins - metabolism; Mercaptoethanol - chemistry; Octoxynol - chemistry; Protein Denaturation; Purification; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; rOmp28; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Triton X-100; β-Mercaptoethanol
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2012.04.002

► Expression of recombinant Omp28 protein of Brucella melitensis in pET28a and pQE30UA vectors. ► Use of 1% Triton X-100 and 20mM β-mercaptoethanol in buffers for single step purification. ► The modified purification protocol yielded 3.6-fold higher rOmp28 protein. ► The modified procedure may be useful for purification of proteins from inclusion bodies. 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.