Nitrogen supplementation ameliorates product quality and quantity during high cell density bioreactor studies of Pichia pastoris : A case study with proteolysis prone streptokinase
Copyright © 2021 Elsevier B.V. All rights reserved..
Streptokinase is a well-established cost-effective therapeutic molecule for thrombo-embolic complications. In the current study, a tag-free variant of streptokinase with a native N-terminus (N-rSK) was developed using the Pichia expression system. A three-copy clone was screened that secreted 1062 mg/L of N-rSK in the complex medium at shake flask level. The biologically active (67,552.61 IU/mg) N-rSK recovered by anion exchange chromatography was predicted to contain 15.43% α-helices, 26.43% β-sheets. The fermentation run in a complex medium yielded a poor quality product due to excessive N-rSK degradation. Therefore, modified basal salt medium was also employed during fermentation operations to reduce the proteolytic processing of the recombinant product. The concomitant feeding of 1 g/L/h soya flour hydrolysate with methanol during the protein synthesis phase reduced the proteolysis and yielded 2.29 g/L of N-rSK. The fermentation medium was also supplemented with urea during growth and induction phases. The combined feeding approach of nitrogen-rich soya flour hydrolysate and urea during bioreactor operations showed significant improvement in protein stability and resulted in a 4-fold increase in N-rSK concentration to a level of 4.03 g/L over shake flask. Under optimized conditions, the volumetric productivity and specific product yield were 52.33 mg/L/h and 33.24 mg/g DCW, respectively.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2021 |
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| Erschienen: |
2021 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:180 |
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| Enthalten in: |
International journal of biological macromolecules - 180(2021) vom: 01. Juni, Seite 760-770 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Adivitiya [VerfasserIn] |
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| Links: |
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| Themen: |
AOX1 promoter |
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| Anmerkungen: |
Date Completed 26.07.2021 Date Revised 26.07.2021 published: Print-Electronic Citation Status MEDLINE |
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| doi: |
10.1016/j.ijbiomac.2021.03.021 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM322729068 |
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| 245 | 1 | 0 | |a Nitrogen supplementation ameliorates product quality and quantity during high cell density bioreactor studies of Pichia pastoris |b A case study with proteolysis prone streptokinase |
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| 500 | |a published: Print-Electronic | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a Copyright © 2021 Elsevier B.V. All rights reserved. | ||
| 520 | |a Streptokinase is a well-established cost-effective therapeutic molecule for thrombo-embolic complications. In the current study, a tag-free variant of streptokinase with a native N-terminus (N-rSK) was developed using the Pichia expression system. A three-copy clone was screened that secreted 1062 mg/L of N-rSK in the complex medium at shake flask level. The biologically active (67,552.61 IU/mg) N-rSK recovered by anion exchange chromatography was predicted to contain 15.43% α-helices, 26.43% β-sheets. The fermentation run in a complex medium yielded a poor quality product due to excessive N-rSK degradation. Therefore, modified basal salt medium was also employed during fermentation operations to reduce the proteolytic processing of the recombinant product. The concomitant feeding of 1 g/L/h soya flour hydrolysate with methanol during the protein synthesis phase reduced the proteolysis and yielded 2.29 g/L of N-rSK. The fermentation medium was also supplemented with urea during growth and induction phases. The combined feeding approach of nitrogen-rich soya flour hydrolysate and urea during bioreactor operations showed significant improvement in protein stability and resulted in a 4-fold increase in N-rSK concentration to a level of 4.03 g/L over shake flask. Under optimized conditions, the volumetric productivity and specific product yield were 52.33 mg/L/h and 33.24 mg/g DCW, respectively | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a AOX1 promoter | |
| 650 | 4 | |a High cell density cultivation | |
| 650 | 4 | |a Nitrogen supplementation | |
| 650 | 4 | |a Pichia pastoris | |
| 650 | 4 | |a Proteolytic degradation | |
| 650 | 4 | |a Streptokinase | |
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| 650 | 7 | |a Streptokinase |2 NLM | |
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| 700 | 1 | |a Khasa, Yogender Pal |e verfasserin |4 aut | |
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