Abstract：
:Production of recombinant proteins is an industrially important technique in the biopharmaceutical sector. Many recombinant proteins are problematic to generate in a soluble form in bacteria as they readily form insoluble inclusion bodies. Recombinant protein solubility can be enhanced by minimising stress imposed on bacteria through decreasing growth temperature and the rate of recombinant protein production. In this study, we determined whether these stress-minimisation techniques can be successfully applied to industrially relevant high cell density Escherichia coli fermentations generating a recombinant protein prone to forming inclusion bodies, CheY-GFP. Flow cytometry was used as a routine technique to rapidly determine bacterial productivity and physiology at the single cell level, enabling determination of culture heterogeneity. We show that stress minimisation can be applied to high cell density fermentations (up to a dry cell weight of >70 g L(-1)) using semi-defined media and glucose or glycerol as carbon sources, and using early or late induction of recombinant protein production, to produce high yields (up to 6 g L(-1)) of aggregation-prone recombinant protein in a soluble form. These results clearly demonstrate that stress minimisation is a viable option for the optimisation of high cell density industrial fermentations for the production of high yields of difficult-to-produce recombinant proteins, and present a workflow for the application of stress-minimisation techniques in a variety of fermentation protocols.
journal_name
J Ind Microbiol Biotechnoljournal_title
Journal of industrial microbiology & biotechnologyauthors
Wyre C,Overton TWdoi
10.1007/s10295-014-1489-1subject
Has Abstractpub_date
2014-09-01 00:00:00pages
1391-404issue
9eissn
1367-5435issn
1476-5535journal_volume
41pub_type
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