In:
Chinese Journal of Agricultural Biotechnology, Cambridge University Press (CUP), Vol. 6, No. 1 ( 2009-04), p. 41-47
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 pK teiR100 . 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 pK teiR100 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 pK teiR100 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 pK teiR100 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.
Type of Medium:
Online Resource
ISSN:
1479-2362
,
1479-2370
DOI:
10.1017/S147923620900254X
Language:
English
Publisher:
Cambridge University Press (CUP)
Publication Date:
2009
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