Structure / function studies of irbp modules expressed as thioredoxin fusion proteins in e. coli

Purpose. To determine the relationship between the quadramodular structure of interphotoreceptor retinoid-binding protein (IRBP) and its function. Methods, A full-length cDNA for IRBP was obtained by screening a Xenopus cDNA library with a partiallength cDNA previously described (Gonzalez-Fernandez et ai, J. Cell Sei. 105:7-21). PCR was used to clone the full-length cDNA, as well as combinations of its modules, into an E. coli thioredoxin expression vector. The expressed proteins were purified by a combination of affinity and classical Chromatographie methods. Purified protein was trypsin digested. The digest was analyzed by capillary LC-electrospray mass spectrometry to measure the molecular weight of the peptides. Sequences for the peptides were determined by collisionally activated dissociation using LC-electrospray-tandem mass spectrometry. Results. Soluble thioredoxin fusion protein was expressed and purified for individual modules of IRBP as well as the full-length protein. Induction of expression at reduced (ITC to 30°C) temperatures was of particular importance in the successful partition of IRBP into the soluble fraction. Conclusions. Thioredoxin fusion technology allows the production of full-length IRBP( 148 kDa with Thioredoxin) and its individual modules as soluble fusion proteins in E. coli. Slowing the rate of expression by reducing temperature may allow E. coli refoldases to keep up with the production of the fusion protein. The authenticity of the recombinant protein can be confirmed by the use of capillary LC-MS and Lctandem mass spectrometry. A binding site for Vitamin A is present in the carboxy terminal module and in the first two N-terminal modules. Production of thioredoxin fusion orotein is an efficient method of oroducine recombinant IRBPs for structure/function studies.