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


RESULTS
The 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 approximately 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.


CONCLUSIONS
We 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.
Krause
Mirja
M
BioSilta Oy, c/o Department of Biochemistry, University of Oulu, Finland.
Ukkonen
Kaisa
K
Haataja
Tatu
T
Ruottinen
Maria
M
Glumoff
Tuomo
T
Neubauer
Antje
A
Neubauer
Peter
P
Vasala
Antti
A
D018533:Biomass; D003470:Culture Media / Q000378:metabolism; D046508:Culture Techniques / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D003470:Culture Media; D011994:Recombinant Proteins; D005947:Glucose; D010100:Oxygen