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Applied and Environmental Microbiology, September 2004, p. 5159-5167, Vol. 70, No. 9
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.9.5159-5167.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Assessment of Production Conditions for Efficient Use of Escherichia coli in High-Yield Heterologous Recombinant Selenoprotein Synthesis

Olle Rengby,¹ Linda Johansson,¹ Lars A. Carlson,¹ Elena Serini,¹ Alexios Vlamis-Gardikas,¹ Per Kårsnäs,² and Elias S. J. Arnér^*

Medical Nobel Institute for Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm,¹ Department of Biology and Chemical Engineering, Mälardalen University, Eskilstuna, Sweden²

Received 7 April 2004/ Accepted 13 May 2004

The production of heterologous selenoproteins in Escherichia coli necessitates the design of a secondary structure in the mRNA forming a selenocysteine insertion sequence (SECIS) element compatible with SelB, the elongation factor for selenocysteine insertion at a predefined UGA codon. SelB competes with release factor 2 (RF2) catalyzing translational termination at UGA. Stoichiometry between mRNA, the SelB elongation factor, and RF2 is thereby important, whereas other expression conditions affecting the yield of recombinant selenoproteins have been poorly assessed. Here we expressed the rat selenoprotein thioredoxin reductase, with titrated levels of the selenoprotein mRNA under diverse growth conditions, with or without cotransformation of the accessory bacterial selA, selB, and selC genes. Titration of the selenoprotein mRNA with a pBAD promoter was performed in both TOP10 and BW27783 cells, which unexpectedly could not improve yield or specific activity compared to that achieved in our prior studies. Guided by principal component analysis, we instead discovered that the most efficient bacterial selenoprotein production conditions were obtained with the high-transcription T7lac-driven pET vector system in presence of the selA, selB, and selC genes, with induction of production at late exponential phase. About 40 mg of rat thioredoxin reductase with 50% selenocysteine content could thereby be produced per liter bacterial culture. These findings clearly illustrate the ability of E. coli to upregulate the selenocysteine incorporation machinery on demand and that this is furthermore strongly augmented in late exponential phase. This study also demonstrates that E. coli can indeed be utilized as cell factories for highly efficient production of heterologous selenoproteins such as rat thioredoxin reductase.

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* Corresponding author. Mailing address: Medical Nobel Institute for Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institute, 171 77 Stockholm, Sweden. Phone: 46-8-5248 69 83. Fax: 46-8-31 15 51. E-mail: Elias.Arner{at}mbb.ki.se.

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Applied and Environmental Microbiology, September 2004, p. 5159-5167, Vol. 70, No. 9
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.9.5159-5167.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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