Expression and characterization of the fourth repeat of Xenopus interphotoreceptor retinoid-binding protein in E. coli.
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 microM). 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 microM. The expression of recombinant IRBP fragments as fusion proteins in prokaryotes will be useful for defining the structural requirements for ligand binding by this interesting protein.
Baer
C A
CA
Department of Ophthalmology, University of Virginia Health Sciences Center, Charlottesville 22908.
Kittredge
K L
KL
Klinger
A L
AL
Briercheck
D M
DM
Braiman
M S
MS
Gonzalez-Fernandez
F
F
D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D005136:Eye Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D008024:Ligands; D017510:Protein Folding; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D012177:Retinol-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014981:Xenopus; D000099214:Retinol-Binding Proteins, Interstitial
D004247:DNA; D005136:Eye Proteins; D008024:Ligands; D011994:Recombinant Proteins; D012177:Retinol-Binding Proteins; D000099214:Retinol-Binding Proteins, Interstitial