Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Process techniques for production of recombinant proteins with Picha pastoris
KTH, Superseded Departments, Biotechnology.
2003 (English)Doctoral thesis, comprehensive summary (Other scientific)
Place, publisher, year, edition, pages
Stockholm: KTH , 2003. , 51 p.
Keyword [en]
p. Pastoris, fed-batch culture, proteolysis, modeling
National Category
Engineering and Technology
Identifiers
URN: urn:nbn:se:kth:diva-3572ISBN: 91-7283-522-2 (print)OAI: oai:DiVA.org:kth-3572DiVA: diva2:9392
Public defence
2003-06-13, 00:00
Note
QC 20100618Available from: 2003-06-26 Created: 2003-06-26 Last updated: 2010-06-18Bibliographically approved
List of papers
1. Modeling of growth and energy metabolism of Pichia pastoris producing a fusion protein
Open this publication in new window or tab >>Modeling of growth and energy metabolism of Pichia pastoris producing a fusion protein
Show others...
2002 (English)In: Bioprocess and biosystems engineering (Print), ISSN 1615-7591, E-ISSN 1615-7605, Vol. 24, no 6, 385-393 p.Article in journal (Refereed) Published
Abstract [en]

A fusion protein composed of a cellulose binding domain from Neocallimastix patriciarum cellulase A and Candida antarctica lipase B (CBD-lipase) was produced by Pichia pastoris methanol utilization plus phenotype in high cell-density cultures. The genes expressing CBD-lipase were fused to the alpha-factor secretion signal sequence of Saccharomyces cerevisiae and placed under the control of the alcohol oxidase gene (AOX1) promoter. To control the repression and induction of AOX1 and oxygen demand at high cell density, a four-stage process was used. Batch growth on glycerol was used in the first step to provide biomass (28 g L-1) while product formation was prevented due to repression of the AOX1. The second stage was exponential fed-batch growth on glycerol, which caused a slight increase of the enzyme alcohol oxidase activity due to derepression of the AOX1. This procedure resulted in smooth transition to exponential fed-batch growth on methanol, the third stage, in which the AOX1 was strongly induced. The fourth stage was constant fed-batch growth on methanol used to control the oxygen demand at the high cell density. A kinetic model was developed that could predict biomass growth and oxygen consumption in processes with and without oxygen-enriched air. With oxygen enrichment to 34% O-2 in the inlet air the methanol feed rate could be increased by 50% and this resulted in 14% higher final cell density (from 140 to 160 g L-1 cell dry weight). The increased methanol feed rate resulted in a proportionally increased specific rate of product secretion to the medium. After an initial decrease, the synthesis capacity of the cell was kept constant throughout the cultivation, which made the product concentration increase almost constantly during the process. The kinetic model also describes how the low maintenance demand of P. pastoris compared with E. coli enables this organism to grow to such high cell densities.

Keyword
ALCOHOL OXIDASE, BATCH, EXPRESSION, YEAST, FERMENTATION, GENES
National Category
Engineering and Technology
Identifiers
urn:nbn:se:kth:diva-13453 (URN)10.1007/s00449-001-0274-5 (DOI)000174810400006 ()
Note
QC 20100618Available from: 2010-06-18 Created: 2010-06-18 Last updated: 2017-12-12Bibliographically approved
2. Analysis and control of proteolysis of a fusion protein in Pichia pastoris fed-batch processes
Open this publication in new window or tab >>Analysis and control of proteolysis of a fusion protein in Pichia pastoris fed-batch processes
Show others...
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.

Keyword
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:nbn:se:kth:diva-13456 (URN)10.1016/S0168-1656(03)00003-8 (DOI)000182299400005 ()
Note
QC 20100618Available from: 2010-06-18 Created: 2010-06-18 Last updated: 2017-12-12Bibliographically approved
3. Production of plant xyloglucan endotransglycosylase (XET) using the methylotrophic yeast Pichia pastoris
Open this publication in new window or tab >>Production of plant xyloglucan endotransglycosylase (XET) using the methylotrophic yeast Pichia pastoris
Show others...
(English)In: Applied Biochemistry and Biotechnology, ISSN 0273-2289, E-ISSN 1559-0291Article in journal (Other academic) Submitted
National Category
Engineering and Technology
Identifiers
urn:nbn:se:kth:diva-13461 (URN)
Note
QC 20100618Available from: 2010-06-18 Created: 2010-06-18 Last updated: 2017-12-12Bibliographically approved
4. Temperature limited fed-batch technique for control of proteolysis in Pichia pastoris bioreactor cultures.
Open this publication in new window or tab >>Temperature limited fed-batch technique for control of proteolysis in Pichia pastoris bioreactor cultures.
Show others...
2003 (English)In: Microbial cell factories, ISSN 1475-2859, Vol. 2, no 1, 6- p.Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: A temperature limited fed-batch (TLFB) technique is described and used for Pichia pastoris Mut+ strain cultures and compared with the traditional methanol limited fed-batch (MLFB) technique. A recombinant fusion protein composed of a cellulose-binding module (CBM) from Neocallimastix patriciarum cellulase 6A and lipase B from Candida antarctica (CALB), was produced and secreted by this strain. RESULTS: A protein concentration of about 1 g L-1 was produced in the MLFB process. However, this product was considerably degraded by protease(s). By applying the TLFB process, the yield was increased to 2 g L-1 full-length product and no proteolytic degradation was observed. Flow cytometry analysis showed that the percentage of dead cells increased rapidly during the initial methanol feed phase in the MLFB process and reached a maximum of about 12% after about 40-70 hours of methanol feeding. In the TLFB process, cell death rate was low and constant and reached 4% dead cells at the end of cultivation (about 150 hours methanol feeding time). The lower cell death rate in the TLFB correlated with a lower protease activity in the culture supernatant. The specific alcohol oxidase (AOX) activity in the TLFB process was 3.5 times higher than in the MLFB process. CONCLUSION: Three mechanisms that may contribute to the much higher accumulation of product in the TLFB process are: 1) reduced proteolysis due to lower temperature, 2) reduced proteolysis due to lower cell death and protease release to the medium, 3) increased synthesis rate due to higher AOX activity.

National Category
Engineering and Technology
Identifiers
urn:nbn:se:kth:diva-13465 (URN)10.1186/1475-2859-2-6 (DOI)12871597 (PubMedID)
Note
QC 20100618Available from: 2010-06-18 Created: 2010-06-18 Last updated: 2010-06-18Bibliographically approved

Open Access in DiVA

fulltext(3820 kB)5072 downloads
File information
File name FULLTEXT01.pdfFile size 3820 kBChecksum MD5
5b2bd6cc949af11af4c1f211e565a3e476d0714fcc5bdd52608ba5695cd33c2a99863cfa
Type fulltextMimetype application/pdf

Search in DiVA

By author/editor
Jahic, Mehmedalija
By organisation
Biotechnology
Engineering and Technology

Search outside of DiVA

GoogleGoogle Scholar
Total: 5072 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 1090 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf