A novel fed-batch based cultivation method provides high cell-density and improves yield of soluble recombinant proteins in shaken cultures

Microbial Cell Factories

Research

Antti Vasala¹  Kaisa Ukkonen¹  Antje Neubauer¹  Maria Ruottinen¹  Mirja Krause²  Tatu Haataja³  Tuomo Glumoff³  Peter Neubauer⁴ 

[1] BioSilta Oy, c/o Department of Biochemistry, University of Oulu, Finland;BioSilta Oy, c/o Department of Biochemistry, University of Oulu, Finland;Fachgebiet Bioverfahrenstechnik, Institut für Biotechnologie, Technische Universität Berlin, Ackerstrasse 71-76, Berlin, Germany;Department of Biochemistry, University of Oulu, Oulu, Finland;Fachgebiet Bioverfahrenstechnik, Institut für Biotechnologie, Technische Universität Berlin, Ackerstrasse 71-76, Berlin, Germany;

关键词: Shake Flask;    High Cell Density;    Mineral Salt Medium;    Recombinant Protein Production;    Shake Flask Culture;   

DOI  :  10.1186/1475-2859-9-11

 received in 2009-11-26, accepted in 2010-02-19,  发布年份 2010

来源: Springer

【 摘 要 】

BackgroundCultivations for recombinant protein production in shake flasks should provide high cell densities, high protein productivity per cell and good protein quality. The methods described in laboratory handbooks often fail to reach these goals due to oxygen depletion, lack of pH control and the necessity to use low induction cell densities. In this article we describe the impact of a novel enzymatically controlled fed-batch cultivation technology on recombinant protein production in Escherichia coli in simple shaken cultures.ResultsThe enzymatic glucose release system together with a well-balanced combination of mineral salts and complex medium additives provided high cell densities, high protein yields and a considerably improved proportion of soluble proteins in harvested cells. The cultivation method consists of three steps: 1) controlled growth by glucose-limited fed-batch to OD600 ~10, 2) addition of growth boosters together with an inducer providing efficient protein synthesis within a 3 to 6 hours period, and 3) a slow growth period (16 to 21 hours) during which the recombinant protein is slowly synthesized and folded. Cell densities corresponding to 10 to 15 g l-1 cell dry weight could be achieved with the developed technique. In comparison to standard cultures in LB, Terrific Broth and mineral salt medium, we typically achieved over 10-fold higher volumetric yields of soluble recombinant proteins.ConclusionsWe have demonstrated that by applying the novel EnBase® Flo cultivation system in shaken cultures high cell densities can be obtained without impairing the productivity per cell. Especially the yield of soluble (correctly folded) proteins was significantly improved in comparison to commonly used LB, Terrific Broth or mineral salt media. This improvement is thought to result from a well controlled physiological state during the whole process. The higher volumetric yields enable the use of lower culture volumes and can thus significantly reduce the amount of time and effort needed for downstream processing or process optimization. We claim that the new cultivation system is widely applicable and, as it is very simple to apply, could widely replace standard shake flask approaches.

【 授权许可】

Unknown   
© Krause et al; licensee BioMed Central Ltd. 2010. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.