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Minimization of aggregation of secreted bivalent anti-human T cell immunotoxin in Pichia pastoris bioreactor culture by optimizing culture conditions for protein secretion

Paper ID Volume ID Publish Year Pages File Format Full-Text
25794 43601 2006 11 PDF Available
Title
Minimization of aggregation of secreted bivalent anti-human T cell immunotoxin in Pichia pastoris bioreactor culture by optimizing culture conditions for protein secretion
Abstract

In a bioreactor culture of genetically engineered Pichia pastoris secreting a bivalent immunotoxin, 64% of the secreted immunotoxin was present in aggregate forms and this resulted in a loss of bioactivity. Biochemical analyses of the secreted immunotoxin and an in vitro aggregation study using purified monomeric immunotoxin suggested that aggregation was primarily an extracellular event. By employing limited methanol feeding at 0.75 ml min−1 per 10 l initial medium, oxygen consumption was reduced, permitting a lowering of the bioreactor agitation speed from 800 to 400 rpm. By increasing the anti-foam reagent to 0.6 ml l−1, the thickness of the air/liquid interfacial foam layer was reduced by 80%. These steps reduced the immunotoxin aggregates from 64% to 5%. Consequently immunotoxin purification yield was increased from 53.0% to 73.8%. Simultaneously this methodology enhanced immunotoxin secretion to 120 mg l−1 at 163 h of methanol induction in a toxin resistant production strain. We conclude that minimizing shearing force and reducing the air/liquid interfacial foam area are crucial factors in reducing hydrophobic protein aggregation upon secretory expression in yeast bioreactor cultures.

Keywords
Pichia pastoris; Protein secretion; Protein aggregation; Recombinant bivalent immunotoxin; Shearing force; Foam formation
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Minimization of aggregation of secreted bivalent anti-human T cell immunotoxin in Pichia pastoris bioreactor culture by optimizing culture conditions for protein secretion
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 121, Issue 1, 2 January 2006, Pages 75–85
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us