Expression and characterization of the fourth repeat of xenopus interphotoreceptor retinoid-binding protein in E. coli

Claxton A. Baer; Karen L. Kittredge; Alexandra L. Klinger; Deborah M. Briercheck; Mark S. Braiman; Federico Gonzalez-Fernandez

doi:10.3109/02713689408999866

Expression and characterization of the fourth repeat of xenopus interphotoreceptor retinoid-binding protein in E. coli

Claxton A. Baer, Karen L. Kittredge, Alexandra L. Klinger, Deborah M. Briercheck, Mark S. Braiman, Federico Gonzalez-Fernandez

Research output: Contribution to journal › Article › peer-review

15 Scopus citations

Abstract

Interphotoreceptor retinoid-binding protein (IRBP) is an extracellular glycolipoprotein which in higher vertebrates has a 4-repeat structure and carries endogenous vitamin A and fatty acids. The location of IRBP's 1-2 binding sites for retinol is unknown. To begin to understand which repeat(s) are responsible for ligand-binding, we expressed the fourth repeat of Xenopus IRBP in E. coli to determine if it could by itself bind all-trans retinol. Our expression studies used a polyhistidine fusion domain to purify the recombinant protein directly from inclusion bodies. The fusion protein could be renatured without aggregation if refolded at a sufficiently dilute concentration (< 3 μM). The recombinant fourth repeat of Xenopus IRBP binds [³H]all-trans retinol and the fluorescence of this ligand increases 8-fold upon binding. The binding is saturable with a K_d = 0.4 μM. The expression of recombinant IRBP fragments as fusion proteins in prokayrotes will be useful for defining the structural requirements for ligand binding by this interesting protein.

Original language	English (US)
Pages (from-to)	391-400
Number of pages	10
Journal	Current Eye Research
Volume	13
Issue number	6
DOIs	https://doi.org/10.3109/02713689408999866
State	Published - 1994
Externally published	Yes

Keywords

Interphotoreceptor retinoid-binding protein
Ligand binding
Retina
Retinoids
Visual cycle
Vitamin A-binding proteins
Xenopus

ASJC Scopus subject areas

Ophthalmology
Sensory Systems
Cellular and Molecular Neuroscience

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@article{473a48d4e7e348209c79f038f9992367,

title = "Expression and characterization of the fourth repeat of xenopus interphotoreceptor retinoid-binding protein in E. coli",

abstract = "Interphotoreceptor retinoid-binding protein (IRBP) is an extracellular glycolipoprotein which in higher vertebrates has a 4-repeat structure and carries endogenous vitamin A and fatty acids. The location of IRBP's 1-2 binding sites for retinol is unknown. To begin to understand which repeat(s) are responsible for ligand-binding, we expressed the fourth repeat of Xenopus IRBP in E. coli to determine if it could by itself bind all-trans retinol. Our expression studies used a polyhistidine fusion domain to purify the recombinant protein directly from inclusion bodies. The fusion protein could be renatured without aggregation if refolded at a sufficiently dilute concentration (< 3 μM). The recombinant fourth repeat of Xenopus IRBP binds [3H]all-trans retinol and the fluorescence of this ligand increases 8-fold upon binding. The binding is saturable with a Kd = 0.4 μM. The expression of recombinant IRBP fragments as fusion proteins in prokayrotes will be useful for defining the structural requirements for ligand binding by this interesting protein.",

keywords = "Interphotoreceptor retinoid-binding protein, Ligand binding, Retina, Retinoids, Visual cycle, Vitamin A-binding proteins, Xenopus",

author = "Baer, {Claxton A.} and Kittredge, {Karen L.} and Klinger, {Alexandra L.} and Briercheck, {Deborah M.} and Braiman, {Mark S.} and Federico Gonzalez-Fernandez",

note = "Funding Information: This work was supported by The Thomas F. Jeffress and Kate Miller Memorial Trust (F.G.-F.); grant #IN149H from the American Cancer Society (F.G.-F.); a student fellowship made in memory of Albert G. and Herman B. Mosler for the Fight for Sight Inc., Research Division of the National Society to Prevent Blindness (C. A.B.); Medical Scientist Training Program of the NIH GM 07267 (C.A.B.); a post-doctoral fellowship supported in part by an award from the Fight for Sight Research Division of the National Society to Prevent Blindness (K.L.K.); an unrestricted developmental grant from Research to Prevent Blindness; a gift from Elizabeth Jones to the University of Virginia for the pediatric eye research; National Science Foundation award DIR-9016 177 for instrumentation and instrument development; National Institutes of Health grant EY094 12 (F. G. -F. ) .",

year = "1994",

doi = "10.3109/02713689408999866",

language = "English (US)",

volume = "13",

pages = "391--400",

journal = "Current Eye Research",

issn = "0271-3683",

publisher = "Taylor and Francis Ltd.",

number = "6",

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T1 - Expression and characterization of the fourth repeat of xenopus interphotoreceptor retinoid-binding protein in E. coli

AU - Baer, Claxton A.

AU - Kittredge, Karen L.

AU - Klinger, Alexandra L.

AU - Briercheck, Deborah M.

AU - Braiman, Mark S.

AU - Gonzalez-Fernandez, Federico

N1 - Funding Information: This work was supported by The Thomas F. Jeffress and Kate Miller Memorial Trust (F.G.-F.); grant #IN149H from the American Cancer Society (F.G.-F.); a student fellowship made in memory of Albert G. and Herman B. Mosler for the Fight for Sight Inc., Research Division of the National Society to Prevent Blindness (C. A.B.); Medical Scientist Training Program of the NIH GM 07267 (C.A.B.); a post-doctoral fellowship supported in part by an award from the Fight for Sight Research Division of the National Society to Prevent Blindness (K.L.K.); an unrestricted developmental grant from Research to Prevent Blindness; a gift from Elizabeth Jones to the University of Virginia for the pediatric eye research; National Science Foundation award DIR-9016 177 for instrumentation and instrument development; National Institutes of Health grant EY094 12 (F. G. -F. ) .

PY - 1994

Y1 - 1994

N2 - Interphotoreceptor retinoid-binding protein (IRBP) is an extracellular glycolipoprotein which in higher vertebrates has a 4-repeat structure and carries endogenous vitamin A and fatty acids. The location of IRBP's 1-2 binding sites for retinol is unknown. To begin to understand which repeat(s) are responsible for ligand-binding, we expressed the fourth repeat of Xenopus IRBP in E. coli to determine if it could by itself bind all-trans retinol. Our expression studies used a polyhistidine fusion domain to purify the recombinant protein directly from inclusion bodies. The fusion protein could be renatured without aggregation if refolded at a sufficiently dilute concentration (< 3 μM). The recombinant fourth repeat of Xenopus IRBP binds [3H]all-trans retinol and the fluorescence of this ligand increases 8-fold upon binding. The binding is saturable with a Kd = 0.4 μM. The expression of recombinant IRBP fragments as fusion proteins in prokayrotes will be useful for defining the structural requirements for ligand binding by this interesting protein.

AB - Interphotoreceptor retinoid-binding protein (IRBP) is an extracellular glycolipoprotein which in higher vertebrates has a 4-repeat structure and carries endogenous vitamin A and fatty acids. The location of IRBP's 1-2 binding sites for retinol is unknown. To begin to understand which repeat(s) are responsible for ligand-binding, we expressed the fourth repeat of Xenopus IRBP in E. coli to determine if it could by itself bind all-trans retinol. Our expression studies used a polyhistidine fusion domain to purify the recombinant protein directly from inclusion bodies. The fusion protein could be renatured without aggregation if refolded at a sufficiently dilute concentration (< 3 μM). The recombinant fourth repeat of Xenopus IRBP binds [3H]all-trans retinol and the fluorescence of this ligand increases 8-fold upon binding. The binding is saturable with a Kd = 0.4 μM. The expression of recombinant IRBP fragments as fusion proteins in prokayrotes will be useful for defining the structural requirements for ligand binding by this interesting protein.

KW - Interphotoreceptor retinoid-binding protein

KW - Ligand binding

KW - Retina

KW - Retinoids

KW - Visual cycle

KW - Vitamin A-binding proteins

KW - Xenopus

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AN - SCOPUS:0028340060

SN - 0271-3683

VL - 13

SP - 391

EP - 400

JO - Current Eye Research

JF - Current Eye Research

IS - 6

ER -

Expression and characterization of the fourth repeat of xenopus interphotoreceptor retinoid-binding protein in E. coli

Abstract

Keywords

ASJC Scopus subject areas

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