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Optimization and efficient purification of recombinant Omp28 protein of Brucella melitensis using Triton X-100 and β-mercaptoethanol.
- Source :
-
Protein expression and purification [Protein Expr Purif] 2012 Jun; Vol. 83 (2), pp. 226-32. Date of Electronic Publication: 2012 Apr 21. - Publication Year :
- 2012
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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.<br /> (Copyright © 2012 Elsevier Inc. All rights reserved.)
- Subjects :
- Bacterial Proteins chemistry
Bacterial Proteins genetics
Bacterial Proteins metabolism
Brucella melitensis metabolism
Cloning, Molecular
Electrophoresis, Polyacrylamide Gel
Escherichia coli
Genetic Vectors
Membrane Proteins chemistry
Membrane Proteins genetics
Membrane Proteins metabolism
Protein Denaturation
Recombinant Proteins chemistry
Recombinant Proteins genetics
Recombinant Proteins metabolism
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Bacterial Proteins isolation & purification
Brucella melitensis genetics
Membrane Proteins isolation & purification
Mercaptoethanol chemistry
Octoxynol chemistry
Recombinant Proteins isolation & purification
Subjects
Details
- Language :
- English
- ISSN :
- 1096-0279
- Volume :
- 83
- Issue :
- 2
- Database :
- MEDLINE
- Journal :
- Protein expression and purification
- Publication Type :
- Academic Journal
- Accession number :
- 22542588
- Full Text :
- https://doi.org/10.1016/j.pep.2012.04.002