Evaluation of disruption methods for the release of intracellular recombinant protein from Escherichia coli for analytical purposes.
The aim of the present study was to find disruption methods that allow fast and reproducible measurement of intracellular recombinant proteins with potential for on-line application. Production of rhSOD (recombinant human superoxide dismutase) by Escherichia coli was used as a model. Three methods of cell disruption, sonication, osmotic shock and chemical treatment using a non-ionic surfactant, were critically compared with respect to efficiency and reproducibility of the release of rhSOD. The release of the recombinant protein was monitored by (i) measurement of the protein content in cell-culture extracts using an SPR (surface plasmon resonance) biosensor, and (ii) assaying the enzyme activity with a colorimetric reagent using a spectrophotometer. Disruption by the non-ionic surfactant showed the best performance in terms of simplicity, reproducibility and efficiency of sample treatment. The surfactant did not interfere with the rhSOD binding to the antibody immobilized on the SPR chip or with the rhSOD activity assay. When comparing the two detection methods during monitoring of an E. coli cultivation, comparable results were obtained.
Division of Biotechnology, Department of Physics and Measurement Technology, Linköping University, SE-581 83 Linköping, Sweden.
Jan
J
Tkac
Igor
I
Vostiar
Carl-Fredrik
CF
Mandenius
D001433:Bacteriolysis / Q000187:drug effects / Q000502:physiology* / Q000528:radiation effects; D002458:Cell Fractionation / Q000379:methods*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000528:radiation effects; D006801:Humans; D009997:Osmotic Pressure; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D013010:Sonication; D013482:Superoxide Dismutase / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D013501:Surface-Active Agents / Q000494:pharmacology*
D011994:Recombinant Proteins; D013501:Surface-Active Agents; D013482:Superoxide Dismutase