Intracellular monitoring of superoxide dismutase expression in an Escherichia coli fed-batch cultivation using on-line disruption with at-line surface plasmon resonance detection.
An on-line cell disruption system for at-line monitoring of the intracellular concentration of recombinant human superoxide dismutase (rhSOD) in a genetically modified Escherichia coli strain, HMS174(DE3) (pET11a/rhSOD), in bioreactor cultivations is described. The sampled bacteria were disrupted on-line by rapid mixing with a nonionic detergent. The recombinant protein content of the lysed bacterial sample was quantitated by a subsequent surface plasmon resonance biosensor with a specific monoclonal antibody. Extraction efficiency of the monitoring system was optimized with respect to the flow rate ratio of the cell suspension and the detergent at relevant cell densities with the aim to attain rapid monitoring. Monitoring was demonstrated for a shake flask culture and a glucose-limited fed-batch cultivation. The results are compared with a traditional enzyme-linked immunosorbent assay method showing a correlation coefficient of R2 = 0.97. Extraction efficiency of rhSOD reached 95-99% at a total processing time of 1.8-2.6 min and a contact time of 0.8-1.4 min. The possibility of extending the monitoring system to other intracellular proteins is discussed.
Vostiar
Igor
I
Division of Biotechnology, IFM, Linköping University, 581 83 Linkoping, Sweden.
Tkac
Jan
J
Mandenius
Carl-Fredrik
CF
D001431:Bacteriological Techniques / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000201:enzymology*; D009862:Online Systems; D013482:Superoxide Dismutase / Q000096:biosynthesis*; D020349:Surface Plasmon Resonance / Q000379:methods*
D013482:Superoxide Dismutase