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Open Access Technical Notes

Expanding the molecular toolbox for Lactococcus lactis: construction of an inducible thioredoxin gene fusion expression system

François P Douillard14, Mary O'Connell-Motherway12, Christian Cambillau3 and Douwe van Sinderen12*

Author Affiliations

1 Department of Microbiology, University College Cork, Cork, Ireland

2 Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland

3 Architecture et Fonction des Macromolécules Biologiques, UMR 6098 Centre National de la Recherche Scientifique and Universités d'Aix-Marseille I & II, Campus de Luminy, Case 932, 13288 Marseille Cedex 09, France

4 Department of Veterinary Sciences, University of Helsinki, Agnes Sjöbergin katu 2, 00790 Helsinki, Finland

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Microbial Cell Factories 2011, 10:66  doi:10.1186/1475-2859-10-66

Published: 9 August 2011

Abstract

Background

The development of the Nisin Inducible Controlled Expression (NICE) system in the food-grade bacterium Lactococcus lactis subsp. cremoris represents a cornerstone in the use of Gram-positive bacterial expression systems for biotechnological purposes. However, proteins that are subjected to such over-expression in L. lactis may suffer from improper folding, inclusion body formation and/or protein degradation, thereby significantly reducing the yield of soluble target protein. Although such drawbacks are not specific to L. lactis, no molecular tools have been developed to prevent or circumvent these recurrent problems of protein expression in L. lactis.

Results

Mimicking thioredoxin gene fusion systems available for E. coli, two nisin-inducible expression vectors were constructed to over-produce various proteins in L. lactis as thioredoxin fusion proteins. In this study, we demonstrate that our novel L. lactis fusion partner expression vectors allow high-level expression of soluble heterologous proteins Tuc2009 ORF40, Bbr_0140 and Tuc2009 BppU/BppL that were previously insoluble or not expressed using existing L. lactis expression vectors. Over-expressed proteins were subsequently purified by Ni-TED affinity chromatography. Intact heterologous proteins were detected by immunoblotting analyses. We also show that the thioredoxin moiety of the purified fusion protein was specifically and efficiently cleaved off by enterokinase treatment.

Conclusions

This study is the first description of a thioredoxin gene fusion expression system, purposely developed to circumvent problems associated with protein over-expression in L. lactis. It was shown to prevent protein insolubility and degradation, allowing sufficient production of soluble proteins for further structural and functional characterization.

Keywords:
nisin; thioredoxin; expression system; Lactococcus lactis