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

Berggren Söderlund, Maria LU ; Johannesson, Gunvor LU and Fex, G (1995) In Biochemical Journal 308(Pt 1). p.353-359
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)],... (More)
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). (Less)
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published
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in
Biochemical Journal
volume
308
issue
Pt 1
pages
353 - 359
publisher
Portland Press Limited
external identifiers
  • pmid:7755585
ISSN
0264-6021
language
English
LU publication?
yes
id
b5efbe63-89f2-4dcd-9686-8a2144367f8a (old id 1109011)
alternative location
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1136884
date added to LUP
2008-07-25 12:08:09
date last changed
2016-04-16 03:13:42
@article{b5efbe63-89f2-4dcd-9686-8a2144367f8a,
  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).},
  author       = {Berggren Söderlund, Maria and Johannesson, Gunvor and Fex, G},
  issn         = {0264-6021},
  language     = {eng},
  number       = {Pt 1},
  pages        = {353--359},
  publisher    = {Portland Press Limited},
  series       = {Biochemical Journal},
  title        = {Expression of human all-trans-retinoic acid receptor beta and its ligand-binding domain in Escherichia coli},
  volume       = {308},
  year         = {1995},
}