Soluble expression in E. coli of a functional interphotoreceptor retinoid-binding protein module fused to thioredoxin: correlation of vitamin A binding regions with conserved domains of C-terminal processing proteases

Experimental Eye Research
C A BaerFederico Gonzalez-Fernandez

Abstract

The exchange of all-trans retinol and 11-cis retinal 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 of IRBP's four exons. Determining the function of the exons has been hampered by insoluble expression of module 4 in Escherichia coli. Here, we found that module 4 of Xenopus IRBP (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-trans retinol 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 a KD=1.25+/-0.39....Continue Reading

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Citations

Sep 8, 2015·Experimental Eye Research·Debashis GhoshFederico Gonzalez-Fernandez
Oct 14, 2003·The Journal of Comparative Neurology·Lisa L Cunningham, Federico Gonzalez-Fernandez
Jun 14, 2000·Experimental Eye Research·M P GeldermanI Gery
Feb 14, 2002·Structure·Andreas Loew, Federico Gonzalez-Fernandez
Nov 13, 2003·Vision Research·Federico Gonzalez-Fernandez
Jan 16, 2014·Experimental Eye Research·Federico Gonzalez-FernandezDebashis Ghosh
Oct 31, 2014·Metallomics : Integrated Biometal Science·Beibei ChenX Chris Le
Nov 27, 2019·Biochimica Et Biophysica Acta. Molecular and Cell Biology of Lipids·Made Airanthi K Widjaja-Adhi, Marcin Golczak

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