Literature DB >> 7755585

Expression of human all-trans-retinoic acid receptor beta and its ligand-binding domain in Escherichia coli.

M Berggren Söderlund1, G Johannesson, G Fex.   

Abstract

all-trans-Retinoic acid, one of the hormonally active derivatives of vitamin A, occurs physiologically in plasma at a concentration below 10 nmol/l. The methods currently used for its quantification are based on HPLC, need about 1 ml of serum, are relatively laborious and thus not well suited for mass analysis. The affinity and specificity of retinoic acid receptors for all-trans-retinoic acid encouraged us to express both the entire human retinoic acid receptor beta (RAR-beta) and two versions of its retinoic acid-binding domain in Escherichia coli in the hope that these recombinant proteins might be used as binders in a ligand-binding assay for all-trans-retinoic acid. The recombinant receptors, the whole receptor [RAR-beta-(V7-Q448)], corresponding to domains A-F, and the ligand-binding domain [RAR-beta-(E149-Q448)], corresponding to domains D-F, were expressed in the vector pET 3d/BL21 (DE3) as inclusion bodies, solubilized with guanidinium chloride, renatured and purified by ion-exchange chromatography. RAR-beta-(P193-Q448), corresponding to domains E-F, was expressed in the vector pET 3d/BL21(DE3)pLysS, and purified by reversed-phase chromatography. Under non-denaturing conditions, the expressed whole receptor [RAR-beta-(V7-Q448)] and the D-F construct (RAR-beta-(E149-Q448)] behaved chromatographically as monomeric proteins whereas the E-F construct [RAR-beta-(P193-Q448)] had a strong tendency to aggregate. RAR-beta-(V7-Q448) and RAR-beta-(E149-Q448) had similar Kd values for all-trans-retinoic acid (1.4 and 0.6 nmol/l respectively) whereas RAR-beta-(P193-Q448) bound all-trans-retinoic acid less avidly (Kd 9.6 nmol/l). 9-cis-Retinoic acid bound to RAR-beta-(E149-Q448) and RAR-beta-(V7-Q448) as avidly as all-trans-retinoic acid. Competition experiments showed weak or no binding of 4-oxo-all-trans-retinoic acid, 4-oxo-13-cis-retinoic acid, 13-cis-retinoic acid, acitretin and retinol by RAR-beta-(E149-Q448).

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Year:  1995        PMID： 7755585      PMCID： PMC1136884     

Source DB:  PubMed          Journal:  Biochem J        ISSN： 0264-6021            Impact factor:   3.857


  23 in total

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Journal:  J Biol Chem       Date:  1991-09-05       Impact factor: 5.157

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Journal:  Eur J Biochem       Date:  1992-03-15

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Journal:  Nature       Date:  1993-11-25       Impact factor: 49.962
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  4 in total

1.  Mouse retinol binding protein gene: cloning, expression and regulation by retinoic acid.

Authors:  K A Jessen; M A Satre
Journal:  Mol Cell Biochem       Date:  2000-08       Impact factor: 3.396

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Authors:  Roberta Galli; Alessio Paone; Muller Fabbri; Nicola Zanesi; Federica Calore; Luciano Cascione; Mario Acunzo; Antonella Stoppacciaro; Andrea Tubaro; Francesca Lovat; Pierluigi Gasparini; Paolo Fadda; Hansjuerg Alder; Stefano Volinia; Antonio Filippini; Elio Ziparo; Anna Riccioli; Carlo M Croce
Journal:  Proc Natl Acad Sci U S A       Date:  2013-05-28       Impact factor: 11.205

3.  Induction of the oxidative catabolism of retinoid acid in MCF-7 cells.

Authors:  M D Krekels; A Verhoeven; J van Dun; W Cools; C Van Hove; L Dillen; M C Coene; W Wouters
Journal:  Br J Cancer       Date:  1997       Impact factor: 7.640

4.  Inhibition of proliferation and induction of apoptosis in soft tissue sarcoma cells by interferon-alpha and retinoids.

Authors:  T Brodowicz; C Wiltschke; D Kandioler-Eckersberger; T W Grunt; M Rudas; S M Schneider; M Hejna; A Budinsky; C C Zielinski
Journal:  Br J Cancer       Date:  1999-07       Impact factor: 7.640
  4 in total

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