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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]
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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

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