2013 ; 23(3):
|Author||Hyun Kang, Micheal B. Ye, Young Yil Bahk|
|Affiliation||Department of Biotechnology College of Biomedical and Health Science, Konkuk University, Chungju-City, 380-701, Korea|
|Title||Expression and Purification of Human Farnesoid X Receptor-Ligand Binding Domain as Soluble Form Using a Dual Cistronic Expression Vector|
J. Microbiol. Biotechnol.2013 ; 23(3):
|Abstract||In this study, we show the expression and purification of
the human recombinant farnesoid X receptor (FXR)-
ligand binding domain (LBD) protein in E. coli using a
double cistronic vector, pACYCDuet-1, as a soluble form.
We describe here the expression and characterization of a
biologically active FXR-LBD(248-476). When expressed in
the influence of bacterial promoters (PT7 and PTac) of the
single cistronic expression vectors, the human recombinant
FXR-LBD(248-476) was found to be totally insoluble. However,
by using a double cistronic expression vector, we were able
to obtain the human recombinant FXR-LBD(248-476) in a
soluble form. To allow for biological activities, we have
subcloned into the pACYCDuet-1 vector, expressed in
E. coli cells at some optimized conditions, and purified and
characterized the human recombinant active FXR-LBD(248-476)
proteins using the fluorescence polarization assay. This
suggests that the expression of FXR-LBD in a double
cistronic vector improves its solubility and probably assists
its correct folding for the biologically active form of the
proteins. We suggest that this may represent a new approach
to high expression of other nuclear receptors and may be
useful as well for other classes of heterodimeric protein
|Keywords||Nuclear receptor, Recombinant FXR-LBD, Dual expression vector, E.coli expression system|
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