Elsevier

Experimental Eye Research

Volume 66, Issue 2, February 1998, Pages 249-262
Experimental Eye Research

Regular article
Soluble Expression inE. coliof a Functional Interphotoreceptor Retinoid-Binding Protein Module Fused to Thioredoxin: Correlation of Vitamin A Binding Regions with Conserved Domains of C-terminal Processing Proteases

https://doi.org/10.1006/exer.1997.0418Get rights and content

Abstract

The exchange of all-transretinol and 11-cisretinal between the photoreceptors and retinal pigmented epithelium is mediated by interphotoreceptor retinoid-binding protein (IRBP). IRBP contains binding sites for retinoids, docosahexaenoic acid and probably cell surface and matrix receptors. IRBP arose through the quadruplication of an ancient protein, represented by its carboxy-terminal module (module 4 in amphibians and mammals). Module 4 has retinol binding activity and is composed of regions coded for by each ofIRBP'sfour exons. Determining the function of the exons has been hampered by insoluble expression of module 4 inEscherichia coli.Here, we found that module 4 ofXenopusIRBP (X4IRBP), as well as its exon segments, can be expressed in a soluble form as thioredoxin fusion proteins. The recombinant proteins were purified by ion exchange and arsenical-based affinity chromatography. Liquid chromatography/mass spectrometry confirmed that the sequence of X4IRBP is correct. All-transretinol binding was characterized by monitoring enhancement of retinol fluorescence, quenching of intrinsic protein fluorescence, and transfer of energy to the bound retinol. Retinol bound to X4IRBP at 2.20±0.29 sites with aKD=1.25±0.39. One of the two sites was localized to Exons(2+3) and had aKD=0.26±0.13 μm. This site, which supported protein quenching and energy transfer, probably contains at least one of the two conserved tryptophans present in this segment. The second site was localized to Exon 4. This site supported the enhancement of retinol fluorescence but not protein quenching or energy transfer and had aKD=1.94±0.20 μm. Exon 1 had no retinol binding activity. The location of the retinol binding regions correlated with the distribution of domains conserved between IRBPs and the newly recognized family of C-terminal processing proteases (CtpAs), proteins which bind and cleave non-polar carboxy termini.

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    Chapman, J. R.

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    Corresponding author: Federico Gonzalez-Fernandez, Department of Ophthalmology Box 475, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, U.S.A.

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