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Fed-batch production of recombinant β-galactosidase using the universal stress promoters uspA and uspB in high cell density cultivations

Lookup NU author(s): Dr Zoltan Pragai, Dr Michael Barer, Professor Colin Harwood

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Abstract

A high-level production system using the universal stress promoters uspA and uspB in a fed-batch cultivation based on minimal medium was designed. In development it was shown that a standard industrial fed-batch protocol could not be used for this purpose since it failed to induce the levels of product as compared to the basal level. Instead, a batch protocol followed by a low constant feed of glucose was shown to give full induction. The levels of the product protein, β-galactosidase, corresponded to approximately 25% of the total protein. Higher levels were found using the uspA than uspB vectors where uspA showed considerably higher basal level. The data indicate that the σ70 regulated promoter, uspA, although affected by the alarmone guanosine tetraphosphate, ppGpp, worked partly in a similar manner to constitutive promoters. An industrial high cell density fed-batch cultivation on the basis of the suggested fed-batch protocol and the uspA promoter gave a final β-galatosidase concentration of 7 g/L and a final cell concentration of 65 g/L. The heterogeneity in production of the individual cell was measured by fluorescence microscopy. The data show that there is a process time independent heterogeneity in production, which is suggested to be caused by heterogeneity in the substrate uptake rate of the individual cell. © 2003 Wiley Periodicals, Inc.


Publication metadata

Author(s): Prytz I, Sanden AM, Nystrom T, Farewell A, Wahlstrom A, Forberg C, Pragai Z, Barer M, Harwood C, Larsson G

Publication type: Article

Publication status: Published

Journal: Biotechnology and Bioengineering

Year: 2003

Volume: 83

Issue: 5

Pages: 595-603

ISSN (print): 0006-3592

ISSN (electronic): 1097-0290

Publisher: John Wiley & Sons, Inc.

URL: http://dx.doi.org/10.1002/bit.10716

DOI: 10.1002/bit.10716

PubMed id: 12827701


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