Analysis and control of proteolysis of a fusion protein in Pichia pastoris fed-batch processes
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
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.
Pichia pastoris, fed-batch cultivation, proteolysis, CELLULOSE-BINDING DOMAIN, CANDIDA-ANTARCTICA, GROWTH-FACTOR, LIPASE-B, YEAST, EXPRESSION, SECRETION, STRAINS
Engineering and Technology
IdentifiersURN: urn:nbn:se:kth:diva-13456DOI: 10.1016/S0168-1656(03)00003-8ISI: 000182299400005OAI: oai:DiVA.org:kth-13456DiVA: diva2:325400
QC 201006182010-06-182010-06-182010-06-18Bibliographically approved