Adaptation of Mycobacterium smegmatis to an Industrial Scale Medium and Isolation of the Mycobacterial PorinMspA
Open Access
- 17 May 2013
- journal article
- Published by Bentham Science Publishers Ltd. in The Open Microbiology Journal
- Vol. 7 (1) , 92-98
Abstract
The adaptation of the organism to a simple and cost-effective growth medium is mandatory in developing a process for large scale production of the octamericporinMspA, which is isolated from Mycobacterium smegmatis. A fermentation optimization with the minimal nutrients required for growth has been performed. During the fermentation, the iron- and ammonium chloride concentrations in the medium were varied to determine their impact on the observed growth rates and cell mass yields. Common antibiotics to control contamination were eliminated in favor of copper sulfate to reduce costs. MspA has been successfully isolated from the harvested M. smegmatisusing aqueous nOPOE (n-octyloligooxyethylene) at 65°C. Because of the extraordinary stability of MspA, it is possible to denature and precipitate virtually all other proteins and contaminants by following this approach. To further purify the product, acetone is used for precipitation. Gel electrophoresis confirmed the presence and purity of MspA. A maximum of 840µg (via Bradford assay) of pure MspA per liter of the optimized simple growth medium has been obtained. This is a 40% increase with respect to the previously reported culture medium for MspA.Keywords
This publication has 21 references indexed in Scilit:
- Role of Porins in Iron Uptake byMycobacterium smegmatisJournal of Bacteriology, 2010
- Poly-N-Isopropyl-acrylamide/Acrylic Acid Copolymers for the Generation of Nanostructures on Mica Surfaces and as Hydrophobic Host Systems for the Porin MspA from Mycobacterium smegmatisThe Journal of Physical Chemistry C, 2009
- Direct Observation of Gold Nanoparticle Assemblies with the Porin MspA on MicaACS Nano, 2009
- Lipid composition and transcriptional response of Mycobacterium tuberculosis grown under iron-limitation in continuous culture: identification of a novel wax esterMicrobiology, 2007
- Nanoarray‐Surfaces by Reconstitution of the Porin MspA into Stabilized Long‐Chain‐Lipid‐Monolayers at a Gold‐SurfaceElectroanalysis, 2006
- Reconstitution of a Porin from Mycobacterium smegmatis at HOPG covered with hydrophobic host layersSurface and Interface Analysis, 2004
- The Core of the Tetrameric Mycobacterial Porin MspA Is an Extremely Stable β-Sheet DomainJournal of Biological Chemistry, 2003
- Nanostructuring by Deposition of Protein Channels Formed on Carbon SurfacesNano Letters, 2002
- Nanostructuring of Carbon Surfaces by Deposition of a Channel-Forming Protein and Subsequent Polymerization of Methyl Methacrylate PrepolymersNano Letters, 2001
- A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye bindingAnalytical Biochemistry, 1976