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Improved production of human type II procollagen in the yeast Pichia pastoris in shake flasks by a wireless-controlled fed-batch system.

Ruottinen M, Bollok M, Kögler M, Neubauer A, Krause M, Hämäläinen ER, Myllyharju J, Vasala A, Neubauer P - BMC Biotechnol. (2008)

Bottom Line: The problem can be solved by applying the fed-batch technology.The presented wireless feeding unit, together with an on-line monitoring system offers a flexible, simple, and low-cost solution for initial optimization of the production in shake flasks which can be performed in parallel.By this way the fed-batch strategy can be applied from the early screening steps also in laboratories which do not have access to high-cost and complicated bioreactor systems.

View Article: PubMed Central - HTML - PubMed

Affiliation: Bioprocess Engineering Laboratory, Dept, of Process and Environmental Engineering, University of Oulu, P.O. Box 4300, FIN-90014 University of Oulu, Finland. maria.ruottinen@aurorabiotec.com

ABSTRACT

Background: Here we describe a new technical solution for optimization of Pichia pastoris shake flask cultures with the example of production of stable human type II collagen. Production of recombinant proteins in P. pastoris is usually performed by controlling gene expression with the strong AOX1 promoter, which is induced by addition of methanol. Optimization of processes using the AOX1 promoter in P. pastoris is generally done in bioreactors by fed-batch fermentation with a controlled continuous addition of methanol for avoiding methanol toxification and carbon/energy starvation. The development of feeding protocols and the study of AOX1-controlled recombinant protein production have been largely made in shake flasks, although shake flasks have very limited possibilities for measurement and control.

Results: By applying on-line pO2 monitoring we demonstrate that the widely used pulse feeding of methanol results in long phases of methanol exhaustion and consequently low expression of AOX1 controlled genes. Furthermore, we provide a solution to apply the fed-batch strategy in shake flasks. The presented solution applies a wireless feeding unit which can be flexibly positioned and allows the use of computer-controlled feeding profiles. By using the human collagen II as an example we show that a quasi-continuous feeding profile, being the simplest way of a fed-batch fermentation, results in a higher production level of human collagen II. Moreover, the product has a higher proteolytic stability compared to control cultures due to the increased expression of human collagen prolyl 4-hydroxylase as monitored by mRNA and protein levels.

Conclusion: The recommended standard protocol for methanol addition in shake flasks using pulse feeding is non-optimal and leads to repeated long phases of methanol starvation. The problem can be solved by applying the fed-batch technology. The presented wireless feeding unit, together with an on-line monitoring system offers a flexible, simple, and low-cost solution for initial optimization of the production in shake flasks which can be performed in parallel. By this way the fed-batch strategy can be applied from the early screening steps also in laboratories which do not have access to high-cost and complicated bioreactor systems.

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SDS-PAGE analysis of expression of human collagen II in P. pastoris shake flask cultivation (experiment 3, Table 1) after 21, 46 and 72 hours cultivation. R represents a reference culture with pulse feeding of methanol and F predetermined quasi-continuous feed. Collagen chains were derived from correctly folded collagen II molecules by HCl-extraction and pepsin digestion. The collagen II chains are marked with an arrow.
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Figure 4: SDS-PAGE analysis of expression of human collagen II in P. pastoris shake flask cultivation (experiment 3, Table 1) after 21, 46 and 72 hours cultivation. R represents a reference culture with pulse feeding of methanol and F predetermined quasi-continuous feed. Collagen chains were derived from correctly folded collagen II molecules by HCl-extraction and pepsin digestion. The collagen II chains are marked with an arrow.

Mentions: All cultures were followed by analysis of collagen II, C-P4H enzymatic activity and product-related mRNAs. The amount of type II collagen chains (139 kD) derived from correctly assembled protease-resistant triple-helical collagen II was analyzed by reducing SDS-PAGE after HCl extraction with pepsin-treatment according to Myllyharju et al. [8]. Stable and correctly assembled collagens are resistant against pepsin. Collagen II showed a clear band (Figure 4) which was significantly stronger in the cultivations with the quasi-continuous feeding profile, compared to the standard pulse feeding profile. The results were reproducible (see Table 1) despite experiment to experiment variations. Generally, more correctly assembled triple-helical collagen was produced in the experiments with quasi-continuous feeding of methanol.


Improved production of human type II procollagen in the yeast Pichia pastoris in shake flasks by a wireless-controlled fed-batch system.

Ruottinen M, Bollok M, Kögler M, Neubauer A, Krause M, Hämäläinen ER, Myllyharju J, Vasala A, Neubauer P - BMC Biotechnol. (2008)

SDS-PAGE analysis of expression of human collagen II in P. pastoris shake flask cultivation (experiment 3, Table 1) after 21, 46 and 72 hours cultivation. R represents a reference culture with pulse feeding of methanol and F predetermined quasi-continuous feed. Collagen chains were derived from correctly folded collagen II molecules by HCl-extraction and pepsin digestion. The collagen II chains are marked with an arrow.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2315644&req=5

Figure 4: SDS-PAGE analysis of expression of human collagen II in P. pastoris shake flask cultivation (experiment 3, Table 1) after 21, 46 and 72 hours cultivation. R represents a reference culture with pulse feeding of methanol and F predetermined quasi-continuous feed. Collagen chains were derived from correctly folded collagen II molecules by HCl-extraction and pepsin digestion. The collagen II chains are marked with an arrow.
Mentions: All cultures were followed by analysis of collagen II, C-P4H enzymatic activity and product-related mRNAs. The amount of type II collagen chains (139 kD) derived from correctly assembled protease-resistant triple-helical collagen II was analyzed by reducing SDS-PAGE after HCl extraction with pepsin-treatment according to Myllyharju et al. [8]. Stable and correctly assembled collagens are resistant against pepsin. Collagen II showed a clear band (Figure 4) which was significantly stronger in the cultivations with the quasi-continuous feeding profile, compared to the standard pulse feeding profile. The results were reproducible (see Table 1) despite experiment to experiment variations. Generally, more correctly assembled triple-helical collagen was produced in the experiments with quasi-continuous feeding of methanol.

Bottom Line: The problem can be solved by applying the fed-batch technology.The presented wireless feeding unit, together with an on-line monitoring system offers a flexible, simple, and low-cost solution for initial optimization of the production in shake flasks which can be performed in parallel.By this way the fed-batch strategy can be applied from the early screening steps also in laboratories which do not have access to high-cost and complicated bioreactor systems.

View Article: PubMed Central - HTML - PubMed

Affiliation: Bioprocess Engineering Laboratory, Dept, of Process and Environmental Engineering, University of Oulu, P.O. Box 4300, FIN-90014 University of Oulu, Finland. maria.ruottinen@aurorabiotec.com

ABSTRACT

Background: Here we describe a new technical solution for optimization of Pichia pastoris shake flask cultures with the example of production of stable human type II collagen. Production of recombinant proteins in P. pastoris is usually performed by controlling gene expression with the strong AOX1 promoter, which is induced by addition of methanol. Optimization of processes using the AOX1 promoter in P. pastoris is generally done in bioreactors by fed-batch fermentation with a controlled continuous addition of methanol for avoiding methanol toxification and carbon/energy starvation. The development of feeding protocols and the study of AOX1-controlled recombinant protein production have been largely made in shake flasks, although shake flasks have very limited possibilities for measurement and control.

Results: By applying on-line pO2 monitoring we demonstrate that the widely used pulse feeding of methanol results in long phases of methanol exhaustion and consequently low expression of AOX1 controlled genes. Furthermore, we provide a solution to apply the fed-batch strategy in shake flasks. The presented solution applies a wireless feeding unit which can be flexibly positioned and allows the use of computer-controlled feeding profiles. By using the human collagen II as an example we show that a quasi-continuous feeding profile, being the simplest way of a fed-batch fermentation, results in a higher production level of human collagen II. Moreover, the product has a higher proteolytic stability compared to control cultures due to the increased expression of human collagen prolyl 4-hydroxylase as monitored by mRNA and protein levels.

Conclusion: The recommended standard protocol for methanol addition in shake flasks using pulse feeding is non-optimal and leads to repeated long phases of methanol starvation. The problem can be solved by applying the fed-batch technology. The presented wireless feeding unit, together with an on-line monitoring system offers a flexible, simple, and low-cost solution for initial optimization of the production in shake flasks which can be performed in parallel. By this way the fed-batch strategy can be applied from the early screening steps also in laboratories which do not have access to high-cost and complicated bioreactor systems.

Show MeSH
Related in: MedlinePlus