Cold-active expression systems
Development of bacterial low-temperature expression systems
A problem of the commercial application of cold-adapted thermosensitive enzymes is their difficult production with the established mesophilic expression systems. These expression systems work well at 37°C. However, at this temperature many cold-adapted enzymes can not correctly fold into their native functional conformation and are either degraded by host proteases or accumulate in form of inclusion bodies. This could hinder an economical production of such enzymes.
We develop cold-active bacterial expression systems, which are suitable for the overproduction of such thermolabile enzymes and other critical proteins. For this purpose we use mainly the Gram-positive bacterium Bacillus subtilis. The application of B. subtilis as expression host is above all preferred, because this bacterium is better adapted to low temperatures, e.g. 20°C, than Escherichia coli. Furthermore, in comparison to E. coli this bacterium has a more efficient secretion apparatus. The gram-positive bacterium B. subtilis is a well known host for the overproduction of technical or pharmaceutical proteins in an industrial scale.
The low-temperature B. subtilis expression systems are based on regulatory sequences of genes coding for cold-shock inducible proteins, especially the cspB- and des-gene. These promoters are highly active at low temperatures.
Process optimization
The genome wide expression analysis of B. subtilis production strains by means of Proteomics or genomic DNA micro arrays enables us to find potential bottle necks in the protein overproduction process.
We verify the quality of recombinant enzymes by means of 2D PAGE and MALDI TOF mass spectrometry (Proteomics).
References
T. KAAN, G. HOMUTH, J. BANDOW and T. SCHWEDER. 2002. Genome-wide transcriptional profiling of the Bacillus subtilis cold shock response. Microbiol. 148: 3441-3455
T. SCHWEDER and G. HOMUTH. 2003. Kälte-induzierbares Expressionssystem. DE10334811.5.
Cooperations
Elisabeth Helmke, Alfred-Wegener-Institut für Polar- und Meeresforschung, Bremerhaven, Germany (http://www.awi-bremerhaven.de/)
Georges Feller, Laboratoire de Biochimie, Université de Liège, Belgium
(http://www.ulg.ac.be/)
Links
How Microbes Beat the Cold: http://www.microbe.org/microbes/psychrophiles.asp
Leben im Eis: http://www.expedition-wasser.de/index00.htm