Assessment of production conditions for efficient use of Escherichia coli in high-yield heterologous recombinant selenoprotein synthesis

Applied and Environmental Microbiology
Olle RengbyElias S J Arnér

Abstract

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 sel...Continue Reading

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Citations

Mar 4, 2014·Archives of Biochemistry and Biophysics·Stephanie Schaefer-RamadanSharon Rozovsky
Jun 22, 2005·Biochimica Et Biophysica Acta·Linda JohanssonElias S J Arnér
Apr 19, 2006·Biochemistry·Brian EckenrothRobert J Hondal
Jan 30, 2009·The Journal of Biological Chemistry·Farnaz Zahedi Avval, Arne Holmgren
Aug 29, 2013·Nucleic Acids Research·Jianqiang XuElias S J Arnér
Apr 3, 2008·PloS One·Karin AnestålElias S J Arnér
Mar 13, 2013·The Journal of Biological Chemistry·Rotem EngelmanMoran Benhar
Sep 22, 2009·Free Radical Biology & Medicine·Sofi E ErikssonElias S J Arnér
May 2, 2009·Biochimica Et Biophysica Acta·Robert J Hondal
Mar 31, 2009·Biochimica Et Biophysica Acta·Satoko Yoshizawa, August Böck
Mar 10, 2009·Experimental Cell Research·Karolina WallenborgOla Hermanson
Jan 17, 2009·Free Radical Biology & Medicine·Olle RengbyElias S J Arnér
Apr 15, 2008·Biochemical and Biophysical Research Communications·Irvens Fanélus, Richard R Desrosiers
Nov 16, 2010·Molecular and Biochemical Parasitology·Amanda L HudsonMary W Davey
Feb 24, 2015·PLoS Neglected Tropical Diseases·Christina A BulmanJudy A Sakanari
Oct 31, 2014·Metallomics : Integrated Biometal Science·Beibei ChenX Chris Le
Feb 27, 2013·International Journal of Molecular Sciences·Ping ChenJia-Zuan Ni
Feb 16, 2018·Science Translational Medicine·William C StaffordElias S J Arnér
Dec 5, 2008·The Journal of Biological Chemistry·Qing ChengElias S J Arnér
Oct 13, 2006·Chembiochem : a European Journal of Chemical Biology·Qing ChengElias S J Arnér

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