Retinol binding protein IV purified from Escherichia coli using intein-mediated cleavage as a suitable replacement for serum sources.

Retinol binding protein IV (RBP) functions as the principal carrier of retinol (Vitamin A) in the blood, where RBP circulates bound to another serum protein, transthyretin. Isolation of pure RBP from the transthyretin complex in human serum can be difficult, but expression of RBP in recombinant systems can circumvent these purification issues. Human recombinant RBP has previously been successfully expressed and purified from E. coli, but recovery of active protein typically requires extensive processing steps, such as denaturing and refolding, and complex purification steps, such as multi-modal chromatography. Furthermore, these methods produce recombinant proteins, often tagged, that display different functional and structural characteristics across systems. In this work, we optimized downstream processing by use of an intein-based expression system in E. coli to produce tag-free, human recombinant RBP (rRBP) with intact native amino termini at yields of up to ~15 mg/L off column. The novel method requires solubilization of inclusion bodies and subsequent oxidative refolding in the presence of retinol, but importantly allows for one-step chromatographic purification that yields high purity rRBP with no N-terminal Met or other tag. Previously reported purification methods typically require two or more chromatographic separation steps to recover tag-free rRBP. Given the interest in mechanistic understanding of RBP transport of retinol in health and disease, we characterized our purified product extensively to confirm rRBP is both structurally and functionally a suitable replacement for serum-derived RBP.

Fatty acid and retinol-binding protein: A novel antigen for immunodiagnosis of human strongyloidiasis.

The tenacious human parasitic helminth Strongyloides stercoralis is a significant health problem worldwide. The current lack of a definitive diagnostic laboratory test to rule out this infection necessitates designing more specific diagnostic methods. Fatty acid and retinol-binding protein (FAR) plays a crucial role in the development and reproduction of nematodes. We generated a recombinant form of this protein and determined its applicability for immunodiagnosis of S. stercoralis. The L3 form of S. stercoralis was harvested and used for RNA extraction and cDNA synthesis. The coding sequence of S. stercoralis FAR (SsFAR) was cloned into pET28a(+) vector, expressed in E. coli BL21 and purified. ELISA and immunoblotting were employed to determine the specificity and sensitivity of rSsFAR using a set of defined sera. In addition, we analyzed the phylogenetic relationship of SsFAR with different FAR sequences from other nematodes. The cloned SsFAR had an open reading frame of 447 bp encoding 147 amino acids, with a deduced molecular mass of 19 kD. The SsFAR amino acid sequence was 93% identical to FAR of S. ratti. For differential immunodiagnosis of strongyloidiasis, rSsFAR exhibited 100% sensitivity and 97% specificity. However, cross-reactivity with FAR proteins of other parasites, namely Toxocara canis and Echinococcus granulosus, was noted. Our results provide a novel approach for immunodiagnosis of S. stercoralis infections using rSsFAR with reliable sensitivity and specificity.

Isolation and characterization of a fatty acid- and retinoid-binding protein from the cereal cyst nematode Heterodera avenae.

A gene encoding fatty acid- and retinoid-binding protein was isolated from the cereal cyst nematode Heterodera avenae and the biochemical function of the protein that it encodes was analysed. The full-length cDNA of the Ha-far-1 gene is 827 bp long and includes a 22- nucleotide trans-spliced leader sequence (SL1) at its 5-end. The genomic clone of Ha-far-1 consists of eight exons separated by seven introns, which range in size from 48 to 186 bp. The Ha-far-1 cDNA contains an open reading frame encoding a 191 amino acid protein, with a predicted secretory signal peptide. Sequence analysis showed that Ha-FAR-1 has highest similarity to the Gp-FAR-1 protein from the potato cyst nematode, Globodera pallida and that the protein was grouped with all homologues from other plant-parasitic nematodes in a phylogenetic analysis. Fluorescence-based ligand binding analysis confirmed that the recombinant Ha-FAR-1 protein was able to bind fatty acids and retinol. Spatial and temporal expression assays showed that the transcripts of Ha-far-1 accumulated mainly in the hypodermis and that the gene is most highly expressed in third-stage juveniles of H. avenae. Fluorescence immunolocalization showed that the Ha-FAR-1 protein was present on the surface of the infective second-stage juveniles of H. avenae. Nematodes treated with dsRNA corresponding to Ha-far-1 showed significantly reduced reproduction compared to nematodes exposed to dsRNA from a non-endogenous gene, suggesting that Ha-far-1 may be an effective target gene for control of H. avenae using an RNAi strategy.

Interphotoreceptor retinoid-binding protein protects retinoids from photodegradation.

Retinol degrades rapidly in light into a variety of photoproducts. It is remarkable that visual cycle retinoids can evade photodegradation as they are exchanged between the photoreceptors, retinal pigment epithelium and Müller glia. Within the interphotoreceptor matrix, all-trans retinol, 11-cis retinol and retinal are bound by interphotoreceptor retinoid-binding protein (IRBP). Apart from its role in retinoid trafficking and targeting, could IRBP have a photoprotective function? HPLC was used to evaluate the ability of IRBP to protect all-trans and 11-cis retinols from photodegradation when exposed to incandescent light (0 to 8842 µW cm(-2)); time periods of 0-60 min, and bIRBP: retinol molar ratios of 1:1 to 1:5. bIRBP afforded a significant prevention of both all-trans and 11-cis retinol to rapid photodegradation. The effect was significant over the entire light intensity range tested, and extended to the bIRBP: retinol ratio 1:5. In view of the continual exposure of the retina to light, and the high oxidative stress in the outer retina, our results suggest IRBP may have an important protective role in the visual cycle by reducing photodegradation of all-trans and 11-cis retinols. This role of IRBP is particularly relevant in the high flux conditions of the cone visual cycle.

Thiol-dependent antioxidant activity of interphotoreceptor retinoid-binding protein.

Interphotoreceptor retinoid-binding protein (IRBP), which is critical to photoreceptor survival and function, is comprised of homologous tandem modules each ∼300 amino acids, and contains 10 cysteines, possibly 8 as free thiols. Purification of IRBP has historically been difficult due to aggregation, denaturation and precipitation. Our observation that reducing agent 1,4-dithiothreitol dramatically prevents aggregation prompted investigation of possible functions for IRBP's free thiols. Bovine IRBP (bIRBP) was purified from retina saline washes by a combination of concanavalin A, ion exchange and size exclusion chromatography. Antioxidant activity of the purified protein was measured by its ability to inhibit oxidation of 2,2'-azinobis [3-ethylbenzothiazoline-6-sulfonate] by metmyoglobin. Homology modeling predicted the relationship of the retinoid binding sites to cysteine residues. As a free radical scavenger, bIRBP was more active than ovalbumin, thioredoxin, and vitamin E analog Trolox. Alkylation of free cysteines by N-ethylmaleimide inhibited bIRBP's antioxidant activity, but not its ability to bind all-trans retinol. Structural modeling predicted that Cys 1051 is at the mouth of the module 4 hydrophobic ligand-binding site. Its free radical scavenging activity points to a new function for IRBP in defining the redox environment in the subretinal space.

Culture of highly differentiated human retinal pigment epithelium for analysis of the polarized uptake, processing, and secretion of retinoids.

The retinal pigment epithelium (RPE) occupies a strategic position within the eye, given its location between the neurosensory retina and the vascular bed (choroid) that nourishes the photoreceptor cells (rods and cones). Among the many attributes of this versatile monolayer of cells is its unique ability to convert vitamin A (retinol) into the prosthetic group (11-cis-retinal) for the rod and cone opsins, the photopigments essential for vision. It does so by absorbing retinol via a receptor-mediated process that involves the interaction of a carrier protein secreted by the liver, retinol-binding protein (RBP), and a receptor/channel that is the gene product of STRA6 (stimulated by retinoic acid 6). Following its uptake through the basolateral plasma membrane of the RPE, retinol encounters a brigade of binding proteins, membrane-bound receptors, and enzymes that mediate its multi-step conversion to 11-cis-retinal and the transport of this visual chromophore to the light-sensitive photoreceptor cell outer segment, the portion of the cell that houses the phototransduction cascade. This process is iterative, repeating itself via the retinoid visual cycle. Most of the human genes that code for this cohort of proteins carry disease-causing mutations in humans. The consequences of these mutations range in severity from relatively mild dysfunction such as congenital stationary night blindness to total blindness. The RPE, although post-mitotic in situ, is capable of proliferation when removed from its native milieu. This offers one the opportunity to study the retinoid visual cycle in modular form, providing insights into this intriguing process in health and disease. This chapter describes a cell culture method whereby the entire visual cycle can be created in vitro.

Assay of retinol-binding protein-transthyretin interaction and techniques to identify competing ligands.

The principles of fluorescence resonance energy transfer have been utilized to develop a high-throughput assay which detects compounds that interfere with interaction between retinol-binding protein (RBP) and transthyretin (TTR). In this assay, the intrinsic fluorescence from the RBP-retinol complex excites a probe molecule which is covalently coupled to TTR. Generation of an emission signal from the TTR probe indicates interaction between RBP-retinol and TTR. Importantly, the inclusion of retinol in the assay allows discrimination of test compounds which bind RBP versus those which bind to TTR. Thus, compounds which bind to RBP must compete with retinol in order to affect RBP-TTR interaction. This feature of the assay will be useful to identify test compounds which are more likely to have an effect in vivo.

The interaction between retinol-binding protein and transthyretin analyzed by fluorescence anisotropy.

The retinol carrier retinol-binding protein (RBP) forms in blood a complex with the thyroid hormone carrier transthyretin (TTR). The interactions of retinoid-RBP complexes, as well as of unliganded RBP, with TTR can be investigated by means of fluorescence anisotropy. RBP represents the prototypic lipocalin, in the internal cavity of which the retinol molecule is accommodated. Due to the tight binding of retinol within a substantially apolar binding site, an intense fluorescence emission characterizes the RBP-bound vitamin. The addition of TTR to the retinol-RBP complex (holoRBP) causes a marked increase in the fluorescence anisotropy of the RBP-bound retinol within the system, due to the formation of the holoRBP-TTR complex, which allows the interaction between the two proteins to be monitored. The fluorescence anisotropy technique is also suitable to study the interaction of TTR with apoRBP and RBP in complex with non-fluorescent retinoids. In the latter cases, the fluorescence signal is provided by a fluorescent probe covalently linked to TTR rather than by RBP-bound retinol. We report here on the preparation of recombinant human RBP and TTR, the covalent labeling of TTR with the fluorescent dansyl probe, and fluorescence anisotropy titrations for RBP and TTR.

Techniques to study specific cell-surface receptor-mediated cellular vitamin A uptake.

STRA6 is a multitransmembrane domain protein that was recently identified as the cell-surface receptor for plasma retinol-binding protein (RBP), the vitamin A carrier protein in the blood. STRA6 binds to RBP with high affinity and mediates cellular uptake of vitamin A from RBP. It is not homologous to any known receptors, transporters, and channels, and it represents a new class of membrane transport protein. Consistent with the diverse physiological functions of vitamin A, STRA6 is widely expressed in diverse adult organs and throughout embryonic development. Mutations in human STRA6 that abolish its vitamin A uptake activity cause severe pathological phenotypes in many human organs including the eye, brain, lung, and heart. This chapter describes functional assays for STRA6 in live cells and on cellular membranes. These assays can be employed to study the mechanism of this new membrane transport mechanism and its roles in the physiology and pathology of many organs.

Purification of the full-length Xenopus interphotoreceptor retinoid binding protein and growth of diffraction-quality crystals.

PURPOSE: Interphotoreceptor retinol-binding protein (IRBP), composed of two or four homologous modules in tandem, plays an important role in retinoid trafficking between the retinal pigmented epithelium, photoreceptors, and Müller cells. The exact nature of this role is not yet clear. Attempts to purify the full-length retinal IRBP to homogeneity for crystallization purposes have largely been unsuccessful, owing primarily to instability and denaturation of the protein at high concentrations in aqueous media. METHODS: A bacterial expression system was used for the production of the recombinant full-length four modules-containing Xenopus IRBP (xIRBP; 1197 amino acids; 131 kDa). An optimized purification strategy and the presence of molar excesses of a thiol-based reducing agent yielded highly pure xIRBP in a soluble, stable and active form, free of its fusion partner. Binding of all-trans retinol was characterized by fluorescence spectroscopy monitoring ligand-fluorescence enhancement, quenching of endogenous protein fluorescence, and energy transfer. RESULTS: We grew the first diffraction-quality crystal of xIRBP. We have gathered diffraction data from these crystals to 2.46 A resolution, sufficient to yield an atomic model of the tertiary structure of IRBP. Retinol-binding results determined by fluorescence spectroscopy show roughly one retinol-binding site per polypeptide chain. CONCLUSIONS: The binding stoichiometry taken together with modeling data suggest that not all modules are functionally equivalent. Determination of the full-length IRBP structure will be a significant breakthrough in understanding the functional roles of IRBP in the visual cycle. The advances presented here will not only lead to the structure of the full-length IRBP, but will allow us to understand how the modules interact in the function of IRBP. Furthermore, these studies will allow characterization of the ligand-binding site(s) with bound ligand(s).

Absence of increased alpha1-microglobulin in IgA nephropathy proteinuria.

To search for biomarkers of IgA nephropathy, protein profiles of urine samples from patients with IgA nephropathy and normal volunteers were compared using two-dimensional DIGE. Most of the 172 spots identified in the urine were serum proteins, and their amounts in IgA nephropathy urine were much higher than those in normal urine; this can be explained as proteinuria caused by glomerular dysfunction. However, only alpha(1)-microglobulin, also one of the major serum proteins, in IgA nephropathy urine was not higher in amount than that in normal urine. We confirmed using ELISA analysis that the amounts of transferrin and albumin in IgA nephropathy and diabetic nephropathy urine were much higher than those in normal urine, whereas the amount of alpha(1)-microglobulin in IgA nephropathy urine was not higher than that in normal urine and was much lower than that in diabetic nephropathy urine. Approximately 50% of alpha(1)-microglobulin forms a complex with IgA in serum. These results suggest that alpha(1)-microglobulin in IgA nephropathy urine is a characteristic protein and might be a biomarker for IgA nephropathy and that alpha(1)-microglobulin might have a relationship with IgA nephropathy pathology.

A system for concomitant overexpression of four periplasmic folding catalysts to improve secretory protein production in Escherichia coli.

Although Escherichia coli is in wide use for preparative protein expression, problems with the folding of the recombinant gene product and protein aggregation are frequently encountered. Apart from cytoplasmic expression, this is also true for secretion into the bacterial periplasm, the method of choice for the production of proteins that carry structural disulfide bonds. Here we report the construction of the helper plasmid pTUM4, which effects overexpression of four established periplasmic chaperones and folding catalysts: the thiol-disulfide oxidoreductases DsbA and DsbC that catalyze the formation and isomerization of disulfide bridges and the peptidyl-prolyl cis/trans-isomerases with chaperone activity, FkpA and SurA. pTUM4 carries a p15a origin of replication and a chloramphenicol resistance gene and, thus, it is compatible with many conventional expression vectors that use the ColEI origin and an ampicillin resistance. Its positive effects on the yield of soluble recombinant protein and the homogeneity of disulfide pattern are illustrated here using the human plasma retinol-binding protein as well as the extracellular carbohydrate recognition domain of the dendritic cell membrane receptor DC-SIGN. Hence, pTUM4 represents a novel helper vector which complements existing cytosolic chaperone coexpression plasmids and should be useful for the functional secretion of various recombinant proteins with hampered folding efficiency.

Improved removal of small proteins using continuous venovenous hemofiltration to treat acute renal failure.

Continuous venovenous hemodialysis (CVVHD) or hemofiltration conducted with pre- (CVVHpre) or post- (CVVHpost) dilution modes are recommended to treat patients with acute renal failure (ARF) and cardiovascular instability. The efficiency of the three techniques was compared in a study including 18 critically ill patients with ARF. Their mean age was 62.1 +/- 16.7 years, and their mean SAPS II score was 59.5 +/- 14.3. They were treated sequentially with the three techniques for periods of 24 hours each (randomized assignment to one technique the first 24 hours followed by the two others). The PRISMA device and M 100 (AN69S) membrane were used in all instances. Blood and replacement (or dialysis) flow rates were kept at 150 and 25 ml/min, respectively. Urea, creatinine, uric acid, inorganic phosphorus, beta2 microglobulin (beta2m), and retinol binding protein (RBP) were measured every 12 hours in plasma and in 12 hours filtrate collection for 3 days. The results are expressed as filtrate/mean plasma (F/P) ratio for the 12 hour period. Removal of small molecules was 16% higher using CVVHD and CVVHpost than CVVHpre. For beta2m and RBP, CVVHpre was, respectively, 43% and 26% more efficient than CVVHD. CVVHpost gave higher but statistically different removal than CVVHpre only for beta2m. CVVHpost was the most efficient technique for removal of small proteins, but this advantage could be easily counterbalanced using higher volume substitution.

A cleavable affinity biotinylating agent reveals a retinoid binding role for RPE65.

Retinal pigment epithelial (RPE) membranes contain the full biochemical apparatus capable of processing all-trans-retinol (vitamin A) into 11-cis-retinal, the visual chromophore. As many of these proteins are integral membrane proteins and resistant to traditional methods of identification, alternate methods of identifying these proteins are sought. The approach described here involves affinity biotinylation with alkali cleavable linkers. A vitamin A containing affinity-labeling haloacetate is described which facilitates the identification of retinoid binding proteins (RBPs). Treatment of crude bovine RPE membranes with (3R)-3-[boc-lys(biotinyl)-O]-all-trans-retinol chloroacetate 1 in the low micromolar range led to the specific labeling of RPE65 and lecithin retinol acyltransferase (LRAT). Only RPE65 is labeled at 5 microM 1 at 4 degrees C. Labeled RPE65 was readily isolated by binding the labeled protein to avidin-containing beads, followed by cleavage of the protein from the beads at pH 11. Trypsin digestion of RPE65 modified by 1, followed by mass spectrometry, demonstrates that C231 and C448 are alkylated by 1. These studies validate the approach that was used, and furthermore demonstrate that RPE65, a major membrane-associated protein of the RPE, is a RBP.

A new method for purification of human plasma retinol-binding protein and transthyretin.

Retinol is transported in the blood bound to a specific carrier protein called retinol-binding protein (RBP), which in turn binds to another protein, transthyretin (TTR), a homotetrameric, thyroid-hormone-transporting protein. Binding of TTR increases the apparent molecular mass of RBP and thereby prevents glomerular filtration of RBP. Owing to their rapid turnover rates, plasma concentrations of these proteins are sensitive indicators and valuable diagnostic markers of vitamin A nutrition, protein energy malnutrition, infection and renal-tubule function. Previously RBP and TTR were purified by using different procedures, either from plasma or from pathological urine of humans. In general the procedure for purification of RBP and TTR is laborious, and extensive sample recycling is necessary for purification in appreciable amounts. In the present study, we have purified RBP using a simple method, which involves (NH(4))(2)SO(4) fractionation followed by sequential gel filtration under native conditions and 6 M urea. TTR, which was eluted in 60 kDa fractions during urea/Sephadex G-100 chromatography, was further purified to homogeneity using a combination of two dye-affinity chromatographic steps on Reactive Yellow and Cibacron Blue coupled to agarose columns. SDS/PAGE and immunoblotting, apart from typical UV absorption and fluorescent properties of RBP, were used for characterizing the purified RBP and TTR. Furthermore, the purified RBP and TTR were found to be functional from mutual binding monitored by fluorescence quenching.

Identification, retinoid binding, and x-ray analysis of a human retinol-binding protein.

Two cellular retinol-binding proteins (CRBP I and II) with distinct tissue distributions and retinoid-binding properties have been recognized thus far in mammals. Here, we report the identification of a human retinol-binding protein resembling type I (55.6% identity) and type II (49.6% identity) CRBPs, but with a unique H residue in the retinoid-binding site and a distinctively different tissue distribution. Additionally, this binding protein (CRBP III) exhibits a remarkable sequence identity (62.2%) with the recently identified iota-crystallin/CRBP of the diurnal gecko Lygodactylus picturatus [Werten, P. J. L., Röll, B., van Alten, D. M. F. & de Jong, W. W. (2000) Proc. Natl. Acad. Sci. USA 97, 3282-3287 (First Published March 21, 2000; 10.1073/pnas.050500597)]. CRBP III and all-trans-retinol form a complex (K(d) approximately 60 nM), the absorption spectrum of which is characterized by the peculiar fine structure typical of the spectra of holo-CRBP I and II. As revealed by a 2.3-A x-ray molecular model of apo-CRBP III, the amino acid residues that line the retinol-binding site in CRBP I and II are positioned nearly identically in the structure of CRBP III. At variance with the human CRBP I and II mRNAs, which are most abundant in ovary and intestine, respectively, the CRBP III mRNA is expressed at the highest levels in kidney and liver thus suggesting a prominent role for human CRBP III as an intracellular mediator of retinol metabolism in these tissues.

Interphotoreceptor retinoid-binding protein contains three retinoid binding sites.

Interphotoreceptor retinoid binding protein (IRBP), the major soluble protein component of the interphotoreceptor matrix, is believed to participate in the visual cycle by transporting retinoids between retinal pigment epithelium and photoreceptor cells in the eye. IRBP can associate with several chemical and isomeric forms of retinoids but displays the highest affinity towards the retinoids that are important in the visual cycle, 11-cis-retinal and all-trans-retinol. It was previously reported that IRBP can associate with 2 mol of all-trans-retinol or 2 mol of 11-cis-retinal per mol of protein. One of the retinoid binding sites, termed 'site 1', was found to display a broad ligand selectivity and to bind either all-trans-retinol or 11-cis-retinal with similar affinities. Here, the retinoid-binding properties of IRBP were further examined. The data demonstrate that IRBP contains three distinct retinoid binding sites. The promiscuous 'site 1', and two additional sites with a stricter selectivity. One of the latter sites appears to be selective towards all-trans-retinol, while the other is specific for 11-cis-retinal.

Thermometric sensing of peroxide in organic media. Application to monitor the stability of RBP-retinol-HRP complex.

The stability of horseradish peroxidase (HRP) in aqueous and organic solvents is applied to develop a simple thermometric procedure to detect the binding of retinoic acid-HRP conjugate to retinol binding protein (RBP). Butanone peroxide (BP) in organic phase and hydrogen peroxide in aqueous phase is detected thermometrically on a HRP column, immobilized by cross-linking with glutaraldehyde on controlled pore glass (CPG). Acetone, acetonitrile, methanol, and 2-butanol are used for detection of BP, in the flow injection analysis (FIA) mode. A linear range between 1 and 50 mM BP is obtained in all the organic solvents with a precision of 5-7% (CV%). The magnitude and nature of the thermometric response is significantly different in each organic solvent. The stability of HRP in the organic phase is used to study the stability of a retinoic acid-HRP conjugate bound to immobilized RBP. The response of HRP (to 20 mM BP) in the retinoic acid-HRP conjugate is used as an indicator of the stability of the RBP-retinoic acid-HRP complex, after challenges with various organic/aqueous solvents. Both immobilized HRP and RBP are stable at least for 6 months. The effect of o-phenylene diamine on the thermometric response of HRP is also investigated. A scheme for the design of a thermometric retinol (vitamin A) biosensor is proposed.

Effects of dispersed point substitutions in Repeat 1 of human interphotoreceptor retinoid binding protein (IRBP).

PURPOSE: The purpose of this study was to measure the effects of mutations on the retinol binding capability of human Repeat 1 of interphotoreceptor retinoid-binding protein (IRBP). First, we predicted important functional amino acids by several computer programs. We also noted the lack of shared functions between Tail-specific protease (Tsp) and IRBP, which bear sequence similarity, and this aided in predicting functional residues. We analyzed the effects of point substitutions on the retinol and fatty acid binding properties of Repeat 1 of human IRBP at 25 and 50 degrees C. METHODS: To find residues critical to retinol binding that might affect function, a series of thirteen mutations were created by site-specific mutagenesis between positions 140 and 280 in Repeat 1 of human IRBP. These mutants were expressed, purified, and tested for binding properties. The conformations of the proteins were examined by circular dichroism (CD) scans. RESULTS: Seven of the mutations exhibited reduced binding capacity, and five were not expressed at high enough levels to assess binding activity. Four of the mutants were purified, and their CD scans were very similar to those of Repeat 1. Only one of the mutations did not affect binding, folding, or expression when compare to wild type Repeat 1. CONCLUSIONS: Several IRBP mutants containing point mutations retained native structure but lost retinol binding function. The data suggest that retinol binding is affected by many different amino acid substitutions in or near a binding pocket. That even a single point substitution can profoundly affect binding without affecting overall conformation suggests that much of Domain B (from amino acid positions 80 to 300) is involved with ligand binding. This excludes three previously proposed IRBP-retinol binding mechanisms: (1) retinol binds to a small portion of the protein repeat, (2) retinol can bind to any hydrophobic patch in the protein, and (3) native conformation is not required for retinol binding to the repeat.

Crystallization and preliminary X-ray data for the human transthyretin-retinol-binding protein (RBP) complex bound to an anti-RBP Fab.

A macromolecular complex of human transthyretin, human retinol-binding protein and an anti-retinol-binding-protein Fab was crystallized by vapour diffusion in sitting drops. Diffraction from these crystals at cryogenic temperatures was consistent with the space group C222, with cell parameters a = 159.34, b = 222.40 and c = 121.27 A. Crystals diffracted to a resolution limit of 3.36 A using synchrotron radiation. Based on a 2:2:1 stoichiometry for the Fab-retinol-binding-protein-transthyretin complex and the presence of one such complex per asymmetric unit, a reasonable Vm coefficient of 2.74 A3 Da-1 could be estimated.