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Evaluation of disruption methods for the release of intracellular recombinant protein from Escherichia coli for analytical purposes
Author(s) -
Tkac Jan,
Vostiar Igor,
Mandenius CarlFredrik
Publication year - 2004
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1042/ba20030143
Subject(s) - escherichia coli , recombinant dna , pulmonary surfactant , surface plasmon resonance , chromatography , sonication , chemistry , intracellular , biosensor , ionic strength , biochemistry , lysis , extracellular , nanotechnology , materials science , aqueous solution , nanoparticle , gene
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.