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Applied and Environmental Microbiology, August 2004, p. 4499-4504, Vol. 70, No. 8
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.8.4499-4504.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Expression of a Temperature-Sensitive Esterase in a Novel Chaperone-Based Escherichia coli Strain

Manuel Ferrer,1* Tatyana N. Chernikova,1,2 Kenneth N. Timmis,1,2 and Peter N. Golyshin1,2

Department of Microbiology, GBF—German Research Centre for Biotechnology, 38124 Braunschweig,1 Institute of Microbiology, Biozentrum, Technical University of Braunschweig, 38106 Braunschweig, Germany2

Received 17 February 2004/ Accepted 14 April 2004

A new principle for expression of heat-sensitive recombinant proteins in Escherichia coli at temperatures close to 4°C was experimentally evaluated. This principle was based on simultaneous expression of the target protein with chaperones (Cpn60 and Cpn10) from a psychrophilic bacterium, Oleispira antarctica RB8T, that allow E. coli to grow at high rates at 4°C (maximum growth rate, 0.28 h–1) (M. Ferrer, T. N. Chernikova, M. Yakimov, P. N. Golyshin, and K. N. Timmis, Nat. Biotechnol. 21:1266-1267, 2003). The expression of a temperature-sensitive esterase in this host at 4 to 10°C yielded enzyme specific activity that was 180-fold higher than the activity purified from the non-chaperonin-producing E. coli strain grown at 37°C (32,380 versus 190 µmol min–1 g–1). We present evidence that the increased specific activity was not due to the low growth temperature per se but was due to the fact that low temperature was beneficial to folding, with or without chaperones. This is the first report of successful use of a chaperone-based E. coli strain to express heat-labile recombinant proteins at temperatures below the theoretical minimum growth temperature of a common E. coli strain (7.5°C).

* Corresponding author. Present address: Institute of Catalysis, CSIC, Campus Cantoblanco, 28049 Madrid, Spain. Phone: 34-91-5854814. Fax: 34-91-5854760. E-mail: mferrer{at}icp.csic.es.

Applied and Environmental Microbiology, August 2004, p. 4499-4504, Vol. 70, No. 8
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.8.4499-4504.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.





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