Purification and functional characterization of human 11beta hydroxylase expressed in Escherichia coli

The FEBS Journal
Andy ZöllnerR Bernhardt

Abstract

The human 11beta-hydroxylase (hCYP11B1) is responsible for the conversion of 11-deoxycortisol into the major mammalian glucocorticoid, cortisol. The reduction equivalents needed for this reaction are provided via a short electron transfer chain consisting of a [2Fe-2S] ferredoxin and a FAD-containing reductase. On the biochemical and biophysical level, little is known about hCYP11B1 because it is very unstable for analyses performed in vitro. This instability is also the reason why it has not been possible to stably express it so far in Escherichia coli and subsequently purify it. In the present study, we report on the successful and reproducible purification of recombinant hCYP11B1 coexpressed with molecular chaperones GroES/GroEL in E. coli. The protein was highly purified to apparent homogeneity, as observed by SDS/PAGE. Upon mass spectrometry, the mass-to-charge ratio (m/z) of the protein was estimated to be 55 761, which is consistent with the value 55 760.76 calculated for the form lacking the translational initiator Met. The functionality of hCYP11B1 was analyzed using different methods (substrate conversion assays, stopped-flow, Biacore). The results clearly demonstrate that the enzyme is capable of hydroxylating its su...Continue Reading

References

Dec 1, 1975·Biochemical and Biophysical Research Communications·S TakemoriM Katagiri
Nov 16, 1992·Biochemical and Biophysical Research Communications·H UhlmannR Bernhardt
Sep 3, 1990·FEBS Letters·T KawamotoY Shizuta
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Jan 1, 1996·Reviews of Physiology, Biochemistry and Pharmacology·R Bernhardt
Feb 24, 2001·Endocrine Research·M HampfR Bernhardt
Dec 18, 2001·Journal of Inorganic Biochemistry·N V BelkinaR Bernhardt
Feb 22, 2002·European Journal of Biochemistry·Stephanie BechtelRita Bernhardt
Nov 13, 2002·Seminars in Reproductive Medicine·Michael Peter
Nov 28, 2002·Biological Chemistry·Matthias BureikRita Bernhardt
Jun 9, 2004·Steroids·Norio KagawaShiro Yoshioka
Jul 17, 2004·The Biochemical Journal·Madanan Madathiparambil GopalakrishnanAndrej Hasilik
Sep 17, 2004·Biochemical and Biophysical Research Communications·Eriko UchidaKuniyo Inouye
Jan 24, 2006·Pharmacology & Therapeutics·Tarek Hakki, Rita Bernhardt
Apr 7, 2006·Biochemical and Biophysical Research Communications·Miharu AraseNorio Kagawa

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Citations

Jul 21, 2010·Applied Microbiology and Biotechnology·Yogan KhatriRita Bernhardt
Jun 16, 2010·Proceedings of the National Academy of Sciences of the United States of America·Alex D SheftelRoland Lill
Jan 29, 2009·Microbial Cell Factories·Olga KolajJ Gerard Wall
Mar 26, 2014·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Lindie Schloms, Amanda C Swart
Mar 16, 2013·Anesthesia and Analgesia·Sivananthaperumal ShanmugasundararajDouglas E Raines
Dec 15, 2015·Drug Metabolism and Disposition : the Biological Fate of Chemicals·Lina SchifferRita Bernhardt
Feb 9, 2016·Expert Opinion on Therapeutic Targets·Rita Bernhardt
Dec 18, 2009·Journal of Molecular Recognition : JMR·Rebecca L Rich, David G Myszka
Aug 26, 2015·The Journal of Steroid Biochemistry and Molecular Biology·Matthew A CernyKosea Frederick
Jan 10, 2017·The Journal of Steroid Biochemistry and Molecular Biology·J NeunzigR Bernhardt
Dec 4, 2014·The Journal of Steroid Biochemistry and Molecular Biology·Lina SchifferRita Bernhardt
Dec 15, 2010·Chembiochem : a European Journal of Chemical Biology·Ming MaZihe Rao
Jan 26, 2011·Anesthesiology·Stuart A Forman
Jun 5, 2014·The FEBS Journal·Elaine W TieuRobert C Tuckey
Mar 21, 2020·Molecular Biology and Evolution·Carrie F Olson-Manning
Jul 31, 2019·The Journal of Steroid Biochemistry and Molecular Biology·Lina SchifferKarl-Heinz Storbeck
Mar 17, 2021·Molecular and Cellular Endocrinology·Rita Bernhardt, Jens Neunzig
Jun 7, 2021·The FEBS Journal·Philip HartzRita Bernhardt
Jul 29, 2009·Biochimica Et Biophysica Acta·Guillermina GoñiMilagros Medina

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