Hostname: page-component-586b7cd67f-r5fsc Total loading time: 0 Render date: 2024-11-24T06:04:05.054Z Has data issue: false hasContentIssue false
  • English
  • Français

Characterization and heterologous expression of testosterone-inducible regulator from Comamonas testosteroni in Escherichia coli

Published online by Cambridge University Press:  24 April 2009

Chen Jian-Qiu ,
Ai Yu-Fang ,
Zhou Yi-Fei ,
Pan Da-Ren ,
Xiong Guang-Ming ,
Guo Yu-Chun ,
Pan Fei  and
Lin Bing-Hui
Chen Jian-Qiu
Affiliation:
College of Crop Science, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, People's Republic of China
Ai Yu-Fang
Affiliation:
College of Life Science, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, People's Republic of China
Zhou Yi-Fei
Affiliation:
College of Crop Science, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, People's Republic of China
Pan Da-Ren*
Affiliation:
College of Life Science, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, People's Republic of China
Xiong Guang-Ming
Affiliation:
Institute of Toxicology and Pharmacology for Natural Scientists, University Medical School Schleswig-Holstein, Campus Kiel, Brunswiker Strasse 10, 24105 Kiel, Germany
Guo Yu-Chun
Affiliation:
College of Crop Science, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, People's Republic of China
Pan Fei
Affiliation:
College of Life Science, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, People's Republic of China
Lin Bing-Hui
Affiliation:
College of Life Science, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, People's Republic of China
*
*Corresponding author. E-mail: [email protected]
Get access Rights & Permissions [Opens in a new window]

Abstract

The testosterone-inducible regulator (teiR) gene was cloned from Comamonas testosteroni chromosomal DNA, and introduced into plasmids pKtac2 (containing a tac promoter) and pK18 to yield plasmids pKtac2-teiR and pKteiR100. The recombinant plasmids were transformed into competent Escherichia coli HB101 and total protein was extracted to detect the TeiR protein expression level using enzyme-linked immunosorbent assay (ELISA). E. coli transformed by pKtac2-teiR and pKteiR100 produced 6.65 and 5.93 μg/mg of TeiR protein, respectively. Recombinant plasmids were also co-transformed into competent E. coli HB101 with plasmid p6 [containing hsdA gene (3α-HSD/CR, 3α-hydroxysteroid dehydrogenase/carbonyl reductase encoding gene)] to reveal the relationship between 3α-HSD/CR and TeiR by ELISA. The amounts of TeiR protein expressed by E. coli containing pKtac2-teiR and pKteiR100 were 5.94 μg/mg and 5.33 μg/mg, respectively, and these increased up to 6.81 μg/mg and 6.10 μg/mg after inducing with 1 mmol/l isopropyl-β-d-thiogalactoside (IPTG). Interestingly, 3α-HSD/CR protein expression level, after co-transformation with plasmids pKtac2-teiR and p6, was lower than that observed in the co-transformation with pKteiR100 and p6. The first co-transformation induced 1.20 μg/mg 3α-HSD/CR protein and the second 1.71 μg/mg. These values rose to 1.42 and 1.80 μg/mg, respectively, after treatment with 1 mmol/l IPTG. Our results proved that the tac promoter was more efficient than the lacZ promoter and that the teiR gene could act as an activator for hsdA gene expression.

Keywords

Comamonas testosteronitestosterone-inducible regulatorELISA3α-hydroxysteroid dehydrogenase/carbonyl reductase
Type
Research Papers
Information
Chinese Journal of Agricultural Biotechnology , Volume 6 , Issue 1 , April 2009 , pp. 41 - 47
Copyright
Copyright © China Agricultural University 2009

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Ausubel, FM, Brent, R, Kingston, RE, et al. (1998) Short Protocols in Molecular Biology, 3rd ed.Beijing: Science Press (in Chinese).Google Scholar
Dai, YM, Zhou, YF, Xiong, G, Wu, MX and Pan, DR (2005) Expression of activator from Comamonas testosteroni with tac promoter containing 10 different consensus sequences. Journal of Agricultural Biotechnology 13(6): 747–753 (in Chinese with English abstract).Google Scholar
Dai, YM, Pan, DR, Xiong, G, Wu, MX and Zhou, YF (2006) Construction and over-expression of the activator gene from Comamonas testosteroni. Journal of Agricultural Biotechnology 14(3): 416–422 (in Chinese with English abstract).Google Scholar
Ducros, VM, Lewis, RJ, Verma, CS, et al. (2001) Crystal structure of GerE, the ultimate transcriptional regulator of spore formation in Bacillus subtilis. Journal of Molecular Biology 306: 759–771.Google Scholar
Egland, KA and Greenberg, EP (2001) Quorum sensing in Vibrio fischeri: analysis of the LuxR DNA binding region by alanine scanning mutagenesis. Journal of Bacteriology 183: 382–386.CrossRefGoogle ScholarPubMed
Horinouchi, M, Hayashi, T, Yamamoto, T and Kudo, T (2003) A new bacterial steroid degradation gene cluster which consists of aromatic compound degradation genes for seco-steroids and 3-ketosteroid dehydrogenase genes in Comamonas testosteroni TA441. Applied and Environmental Microbiology 69: 4421–4430.CrossRefGoogle ScholarPubMed
Horinouchi, M, Kurita, T, Yamamoto, T, Hatori, E, Hayashi, T and Kudo, T (2004a) Steroid degradation gene cluster of Comamonas testosteroni consisting of 18 putative genes from meta-cleavage enzyme gene tesB to regulator gene tesR. Biochemical and Biophysical Research Communications 324: 597–604.CrossRefGoogle ScholarPubMed
Horinouchi, M, Hayashi, T and Kudo, T (2004b) The genes encoding the hydroxylase of 3-hydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione in steroid degradation in Comamonas testosteroni TA441. Journal of Steroid Biochemistry and Molecular Biology 92: 143–154.CrossRefGoogle ScholarPubMed
Horinouchi, M, Hayashi, T, Koshino, H, Kurita, T and Kudo, T (2005) Identification of 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid, 4-hydroxy-2-oxohexanoic acid, and 2-hydroxyhexa-2,4-dienoic acid and related enzymes involved in testosterone degradation in Comamonas testosteroni TA441. Applied and Environmental Microbiology 71: 5275–5281.Google Scholar
Horinouchi, M, Hayashi, T, Koshino, H and Kudo, T (2006) ORF18-disrupted mutant of Comamonas testosteroni TA441 accumulates significant amounts of 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid and its derivatives after incubation with steroids. Journal of Steroid Biochemistry and Molecular Biology 101: 78–84.CrossRefGoogle ScholarPubMed
Leppik, RA (1983) Deoxycholic acid degradation by a Pseudomonas sp.: acidic intermediates with A-ring unsaturation. Biochemical Journal 210: 829–836.CrossRefGoogle ScholarPubMed
Otto, CM, Niagro, F, Su, X and Rawlings, CA (1995) Expression of recombinant feline tumor necrosis factor is toxic to Escherichia coli. Clinical and Diagnostic Laboratory Immunology 2: 740–746.CrossRefGoogle ScholarPubMed
Pristovsek, P, Sengupta, K, Lohr, F, et al. (2003) Structural analysis of the DNA-binding domain of the Erwinia amylovora RcsB protein and its interaction with the RcsAB box. Journal of Biological Chemistry 278: 17752–17759.CrossRefGoogle ScholarPubMed
Pruneda-Paz, JL, Linares, M, Cabrera, JE and Raimondi, SG (2004) TeiR, a LuxR-type transcription factor required for testosterone degradation in Comamonas testosteroni. Journal of Bacteriology 186(5): 1430–1437.CrossRefGoogle ScholarPubMed
Sambrook, J and Russell, DW (2002) Molecular Cloning: A Laboratory Manual, 3rd ed.Beijing: Science Press (in Chinese).Google Scholar
Vannini, A, Volpari, C, Gargioli, C, et al. (2002) The crystal structure of the quorum sensing protein TraR bound to its autoinducer and target DNA. EMBO Journal 21: 4393–4401.Google Scholar
Xiong, G, Martin, HJ and Maser, E (2003a) Characterization and recombinant expression of the translational repressor RepB of 3α-hydroxysteroid dehydrogenase/carbonyl reductase in Comamonas testosteroni. Chemico-Biological Interactions 143(144): 425–433.CrossRefGoogle ScholarPubMed
Xiong, G, Martin, HJ and Maser, E (2003b) Identification and characterization of a translational repressor of the steroid-inducible 3α-hydroxysteroid dehydrogenase/carbonyl reductase gene in Comammonas testeroni. Journal of Biological Chemistry 278(48): 47400–47407.Google Scholar