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Analysis and control of proteolysis of a fusion protein in Pichia pastoris fed-batch processes
KTH, Superseded Departments, Biotechnology.
KTH, Superseded Departments, Biotechnology.
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2003 (English)In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 102, no 1, 45-53 p.Article in journal (Refereed) Published
Abstract [en]

A fusion protein composed of a cellulose-binding module (CBM) from Neocallimastix patriciarum cellulase 6A and lipase B from Candida antarctica (CALB), was produced by Pichia pastoris Mut(+) in high-cell density bioreactor cultures. The production was induced by switching from growth on glycerol to growth on methanol. The lipase activity in the culture supernatant increased at an almost constant rate up to a value corresponding to 1.3 g l(-1) of CBM-CALB. However, only about 40% of the product was of full-length according to Western blot analysis. This loss was due to a cleavage of the protein in the linker between the CBM and the CALB moieties. The cleavage was catalyzed by serine proteases in the culture supernatant. The CALB-moiety was subjected to further slow degradation by cell-associated proteolysis. Different strategies were used to reduce the proteolysis. Previous efforts to shorten the linker region resulted in a stable protein but with ten times reduced product concentration in bioreactor cultures (Gustavsson et al. 2001, Protein Eng. 14, 711-715). Addition of rich medium for protease substrate competition had no effect on the proteolysis of CBM-CALB. The kinetics for the proteolytic reactions, with and without presence of cells were shown to be influenced by pH. The fastest reaction, cleavage in the linker, was substantially reduced at pH values below 5.0. Decreasing the pH from 5.0 to 4.0 in bioreactor cultures resulted in an increase of the fraction of full-length product from 40 to 90%. Further improvement was achieved by decreasing the temperature from 30 to 22 degreesC during the methanol feed phase. By combining the optimal pH and the low temperature almost all product (1.5 g l(-1)) was obtained as full-length protein with a considerably higher purity in the culture supernatant compared with the original cultivation.

Place, publisher, year, edition, pages
2003. Vol. 102, no 1, 45-53 p.
Keyword [en]
Pichia pastoris, fed-batch cultivation, proteolysis, CELLULOSE-BINDING DOMAIN, CANDIDA-ANTARCTICA, GROWTH-FACTOR, LIPASE-B, YEAST, EXPRESSION, SECRETION, STRAINS
National Category
Engineering and Technology
Identifiers
URN: urn:nbn:se:kth:diva-13456DOI: 10.1016/S0168-1656(03)00003-8ISI: 000182299400005OAI: oai:DiVA.org:kth-13456DiVA: diva2:325400
Note
QC 20100618Available from: 2010-06-18 Created: 2010-06-18 Last updated: 2017-12-12Bibliographically approved
In thesis
1. Process techniques for production of recombinant proteins with Picha pastoris
Open this publication in new window or tab >>Process techniques for production of recombinant proteins with Picha pastoris
2003 (English)Doctoral thesis, comprehensive summary (Other scientific)
Place, publisher, year, edition, pages
Stockholm: KTH, 2003. 51 p.
Keyword
p. Pastoris, fed-batch culture, proteolysis, modeling
National Category
Engineering and Technology
Identifiers
urn:nbn:se:kth:diva-3572 (URN)91-7283-522-2 (ISBN)
Public defence
2003-06-13, 00:00
Note
QC 20100618Available from: 2003-06-26 Created: 2003-06-26 Last updated: 2010-06-18Bibliographically approved

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