High-level expression of recombinant human serum albumin from the methylotrophic yeast Pichia pastoris with minimal protease production and activation

Kobayashi, Kaoru; Kuwae, Shinobu; Ohya, Takeshi; Ohda, Toyoo; Ohyama, Masao; Ohi, Hideyuki; Tomomitsu, Kenji; Ohmura, Takao

doi:10.1016/s1389-1723(00)88050-0

Cited by 177 publications

(119 citation statements)

References 15 publications

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“…The fermentation stage after 24 hours was a high oxygen utilization process, and DO level dropped rapidly during this period to zero at lower agitation rate. However, the DO level at high agitation rate of 750 rpm was always higher than the critical value of 10 % for the microorganism reported by Kobayashi et al (2000).…”

Section: Conventionally Sparged Fermentation At 750 Rpmcontrasting

confidence: 56%

“…At this very high agitation rate, a high cell density of 138.2 g / L was achieved due to sufficient oxygen dispersion in the fermentation medium ( Kobayashi et al (2000). The improved protein production was also attributed to the more efficient oxygen delivery to the microorganisms.…”

Section: Conventionally Sparged Fermentation At 750 Rpmmentioning

confidence: 96%

“…LITERATURE REVIEW Bovine lysozyme S 0.30 Digan et al 1989 Human serum albumin (HSA) S 1.40 Kobayashi et al 2000 Human epidermal growth factor S 0.50 Siegel et al 1990 Mouse epidermal growth factor S 0.45 Aprotinin analog S 0.80 Vedvick et al 1991 Kunitz protease inhibitor S 1.0 Wagner et al 1992 + I = Intracellular, S = Secreted.…”

Section: Human Serum Albuminmentioning

confidence: 99%

“…Fed-batch fermentations of P. pastoris for rHSA production were conducted in a 3-L fermenter (Kobayashi et al, 2000). Maximum dry cell weight was around 80 g / L and rHSA production reached 360 mg / L at 96.5 h of cultivation.…”

Section: Human Serum Albuminmentioning

confidence: 99%

“…Kobayashi et al (2000) reported that the protein production reached its maximum at 96.5 h of cultivation. To determine the optimal fermentation length so that maximal amount of protein could be obtained without further incubation, 121 hours of conventionally sparged fermentation was conducted in the 1.6-L Bioflo III fermenter at agitation rate of 350 rpm.…”

Section: Conventionally Sparged Fermentationsmentioning

confidence: 99%

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Microbubble fermentation of recombinant Pichia pastoris for human serum albumin production

Zhang¹,

Li²,

Agblevor³

2005

Process Biochemistry

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The high cell density fermentation of recombinant Pichia pastoris for human serum albumin (HSA) production is a high oxygen demand process. The oxygen demand is usually met by increased agitation rate and use of oxygen-enriched air. Microbubble fermentation however can supply adequate oxygen to the microorganisms at relatively low agitation rates because of improved mass transfer of the microbubbles used for the sparging. Conventionally sparged fermentations were conducted for the production of HSA using P. pastoris at agitation rates of 350, 500, and 750 rpm, and were compared to MBD sparged fermentation at 150, 350, and 500 rpm agitation rates. The MBD improved the volumetric oxygen transfer coefficient (k L a) and subsequently increased the cell mass and protein production compared to conventional fermentation.Cell production in MBD fermentation at 350 rpm was 4.6 times higher than that in the conventional fermentation at 350 rpm, but similar to that in the conventional 750 rpm. The volumetric oxygen transfer coefficient k L a was 1011.9 h -1 in MBD 350 rpm, which was 6.1 times higher than that in the conventional 350 rpm (164.9 h -1 ) but was similar to the conventional 750 rpm (1098 h -1 ). Therefore, MBD fermentation results at low agitation of 350 rpm were similar to those in the conventional fermentation at high agitation of 750 rpm. There was considerable improvement in oxygen transfer to the microorganism using MBD sparging relative to the conventional sparging.Conventional fermentations were conducted both in a Biostat Q fermenter (small) at 500 rpm, 750 rpm, and 1000 rpm, and in a Bioflo III fermenter (large) at 350 rpm, 500 rpm, and 750 rpm. At the same agitation rate of 500 rpm, cell production in the large reactor was 3.8 times higher than that in the small one, and no detectable protein was produced in the small reactor at 500 rpm. At the same agitation rate of 750 rpm, both cell production and protein production in the large reactor were 4.6 times higher than the small reactor. Thus, the Bioflo III fermenter showed higher oxygen transfer efficiency than the Biostat Q fermenter, because of the more efficient aeration design of the Bioflo III fermenter. ACKNOWLEDGMENTS

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Section: Conventionally Sparged Fermentation At 750 Rpmcontrasting

confidence: 56%

Section: Conventionally Sparged Fermentation At 750 Rpmmentioning

confidence: 96%

Section: Human Serum Albuminmentioning

confidence: 99%

Section: Human Serum Albuminmentioning

confidence: 99%

Section: Conventionally Sparged Fermentationsmentioning

confidence: 99%

See 3 more Smart Citations

Microbubble fermentation of recombinant Pichia pastoris for human serum albumin production

Zhang¹,

Li²,

Agblevor³

2005

Process Biochemistry

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Transgenic Rice for the Production of Recombinant Pharmaceutical Proteins

Yang

Shi

et al. 2018

Molecular Pharming

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RSM optimization of HSA/IL1Ra in Pichia pastoris overexpression strain and study of its in vivo activity in reducing hyperglycemia of GK rats

Liu

Zhao

Lai

et al. 2017

Biotech and App Biochem

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Human serum albumin (HSA) and interleukin-1 receptor antagonist (IL1Ra) fusion protein is a potential long-acting drug in the treatment of type 2 diabetes. Previously, the expression level of HSA/IL1Ra in Pichia pastoris was successfully improved by increasing the gene copy number and coexpression with chaperone (protein disulfide isomerase) in our laboratory. However, the overexpression strain resulted in low production of high- cell-density fermentation. In this study, the culture medium was optimized in both flask and fermenter, and the optimum culture medium notably increased the productivity and stability of HSA/IL1Ra. To further improve the expression, response surface methodology was used to further optimize the culture condition through modeling three selected parameters (induction pH, induction temperature [T], and maximum methanol feed rate [V ]). The maximum yield of HSA/IL1Ra reached 1.1 g/L (10-fold higher than original fermentation condition) under the optimized culture condition (pH 7.0, T = 29 ℃ and V = 4.82 mL/L/H) in a 5-L fermenter. In addition, the degradation position of HSA/IL1Ra during fermentation was determined to be K571, serving as a potential target for genetic modification strategies to reduce the degradation. Finally, the in vivo activity study showed that HSA/IL1Ra maintained the therapeutic effect of IL1Ra in type 2 diabetes model rats meanwhile reducing the frequency of administration.

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High-level expression of recombinant human serum albumin from the methylotrophic yeast Pichia pastoris with minimal protease production and activation

Cited by 177 publications

References 15 publications

Microbubble fermentation of recombinant Pichia pastoris for human serum albumin production

Microbubble fermentation of recombinant Pichia pastoris for human serum albumin production

Transgenic Rice for the Production of Recombinant Pharmaceutical Proteins

RSM optimization of HSA/IL1Ra in Pichia pastoris overexpression strain and study of its in vivo activity in reducing hyperglycemia of GK rats

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