Fed-batch production of recombinant beta-galactosidase using the universal stress promoters uspA and uspB in high cell density cultivations
2003 (English)In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 83, no 5, 595-603 p.Article in journal (Refereed) Published
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, beta-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 sigma(70) 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 fedbatch cultivation on the basis of the suggested fed-batch protocol and the uspA promoter gave a final beta-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.
Place, publisher, year, edition, pages
2003. Vol. 83, no 5, 595-603 p.
stress promoters, uspA, uspB, recombinant protein production, fed-batch, high cell density, plasmid and chromosomal construction, escherichia-coli, protein expression, limit growth, genes, strategies, starvation, cloning
Biochemistry and Molecular Biology
IdentifiersURN: urn:nbn:se:kth:diva-22690DOI: 10.1002/bit.10716ISI: 000184310700010OAI: oai:DiVA.org:kth-22690DiVA: diva2:341388
QC 201005252010-08-102010-08-102010-10-08Bibliographically approved