Please use this identifier to cite or link to this item: http://dx.doi.org/10.14279/depositonce-4551
Main Title: Effect of culture medium, host strain and oxygen transfer on recombinant Fab antibody fragment yield and leakage to medium in shaken E. coli cultures
Author(s): Ukkonen, Kaisa
Veijola, Johanna
Vasala, Antti
Neubauer, Peter
Type: Article
Language: English
Language Code: en
Abstract: Background: Fab antibody fragments in E. coli are usually directed to the oxidizing periplasmic space for correct folding. From periplasm Fab fragments may further leak into extracellular medium. Information on the cultivation parameters affecting this leakage is scarce, and the unpredictable nature of Fab leakage is problematic regarding consistent product recovery. To elucidate the effects of cultivation conditions, we investigated Fab expression and accumulation into either periplasm or medium in E. coli K-12 and E. coli BL21 when grown in different types of media and under different aeration conditions. Results: Small-scale Fab expression demonstrated significant differences in yield and ratio of periplasmic to extracellular Fab between different culture media and host strains. Expression in a medium with fed-batch-like glucose feeding provided highest total and extracellular yields in both strains. Unexpectedly, cultivation in baffled shake flasks at 150 rpm shaking speed resulted in higher yield and accumulation of Fabs into culture medium as compared to cultivation at 250 rpm. In the fed-batch medium, extracellular fraction in E. coli K-12 increased from 2-17% of total Fab at 250 rpm up to 75% at 150 rpm. This was partly due to increased lysis, but also leakage from intact cells increased at the lower shaking speed. Total Fab yield in E. coli BL21 in glycerol-based autoinduction medium was 5 to 9-fold higher at the lower shaking speed, and the extracellular fraction increased from ≤ 10% to 20-90%. The effect of aeration on Fab localization was reproduced in multiwell plate by variation of culture volume. Conclusions: Yield and leakage of Fab fragments are dependent on expression strain, culture medium, aeration rate, and the combination of these parameters. Maximum productivity in fed-batch-like conditions and in autoinduction medium is achieved under sufficiently oxygen-limited conditions, and lower aeration also promotes increased Fab accumulation into extracellular medium. These findings have practical implications for screening applications and small-scale Fab production, and highlight the importance of maintaining consistent aeration conditions during scale-up to avoid changes in product yield and localization. On the other hand, the dependency of Fab leakage on cultivation conditions provides a practical way to manipulate Fab localization.
URI: urn:nbn:de:kobv:83-opus4-68757
http://depositonce.tu-berlin.de/handle/11303/4848
http://dx.doi.org/10.14279/depositonce-4551
Issue Date: 2013
Date Available: 17-Jul-2015
DDC Class: 570 Biowissenschaften; Biologie
610 Medizin und Gesundheit
Subject(s): Autoinduktion
Fab-Fragment
Fed-batch-Verfahren
Periplasmatische Expression
Sauerstoffzufuhr
Autoinduction
Fab fragment
Fed-batch
Oxygen transfer
Periplasmic expression
Creative Commons License: https://creativecommons.org/licenses/by/2.0/
Journal Title: Microbial Cell Factories
Publisher: BioMed Central
Publisher Place: London
Volume: 12
Article Number: 73
Publisher DOI: 10.1186/1475-2859-12-73
Notes: First published by BioMed Central Ukkonen, Kaisa ; Veijola, Johanna ; Vasala, Antti ; Neubauer, Peter : Effect of culture medium, host strain and oxygen transfer on recombinant Fab antibody fragment yield and leakage to medium in shaken E. coli cultures. - In: Microbial Cell Factories. - ISSN 1475-2859 (online). - 12 (2013), art. 73. - doi:10.1186/1475-2859-12-73.
Appears in Collections:Technische Universität Berlin » Fakultäten & Zentralinstitute » Fakultät 3 Prozesswissenschaften » Institut für Biotechnologie » Publications

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