Picomolar detection of retinol binding protein 4 for early management of type II diabetes.

Type (II) diabetes is one of the major threats to mankind as it causes insulin resistance in human body and Retinol Binding Protein 4 (RBP4) is currently considered as a potential biomarker for early management of this disease. Hence a low-level detection of RBP4 is a very important task and for this purpose, a novel RBP4 biosensor has been developed using homemade plastic chip electrodes (PCEs) as a platform for self-assembled monolayer (SAM) of 4-ATP and further functionalization with glutaraldehyde. Anti RBP4 is used as biorecognition species and electrochemical impedance spectroscopy has been performed to detect different RBP4 concentrations plotted against charge transfer resistance. A wide concentration range from 100 fg/mL to 1 ng/mL has been tested and a low limit of detection (LOD) of 100 fg/mL has been achieved. This is the first report for fabrication of electrochemical biosensor of RBP4 using Ag-Ab interaction having such low LOD. The sensor is characterized by various physico-chemical techniques. Excellent reproducibility and quick measurement make this biosensor extremely useful for the biomedical industry.

Human plasma retinol-binding protein (RBP4) is also a fatty acid-binding protein.

RBP4 (plasma retinol-binding protein) is the 21 kDa transporter of all-trans retinol that circulates in plasma as a moderately tight 1:1 molar complex of the vitamin with the protein. RBP4 is primarily synthesized in the liver but is also produced by adipose tissue and circulates bound to a larger protein, transthyretin, TTR, that serves to increase its molecular mass and thus avoid its elimination by glomerular filtration. This paper reports the high resolution three-dimensional structures of human RBP4 naturally lacking bound retinol purified from plasma, urine and amniotic fluid. In all these crystals we found a fatty acid molecule bound in the hydrophobic ligand-binding site, a result confirmed by mass spectrometry measurements. In addition we also report the 1.5 Šresolution structures of human holo-RBP4 and of the protein saturated with palmitic and lauric acid and discuss the interaction of the fatty acids and retinol with the protein.

Real-time analyses of retinol transport by the membrane receptor of plasma retinol binding protein.

Vitamin A is essential for vision and the growth/differentiation of almost all human organs. Plasma retinol binding protein (RBP) is the principle and specific carrier of vitamin A in the blood. Here we describe an optimized technique to produce and purify holo-RBP and two real-time monitoring techniques to study the transport of vitamin A by the high-affinity RBP receptor STRA6. The first technique makes it possible to produce a large quantity of high quality holo-RBP (100%-loaded with retinol) for vitamin A transport assays. High quality RBP is essential for functional assays because misfolded RBP releases vitamin A readily and bacterial contamination in RBP preparation can cause artifacts. Real-time monitoring techniques like electrophysiology have made critical contributions to the studies of membrane transport. The RBP receptor-mediated retinol transport has not been analyzed in real time until recently. The second technique described here is the real-time analysis of STRA6-catalyzed retinol release or loading. The third technique is real-time analysis of STRA6-catalyzed retinol transport from holo-RBP to cellular retinol binding protein I (CRBP-I). These techniques provide high sensitivity and resolution in revealing RBP receptor's vitamin A uptake mechanism.

Expression and characterization of recombinant human retinol-binding protein in Pichia pastoris.

Plasma retinol-binding protein (RBP4) is the principal carrier of vitamin A in blood. Recent studies have suggested that RBP4 may have also a role in insulin resistance. To date the recombinant protein is usually produced by refolding inclusion bodies in Escherichia coli. Here we report the expression and characterization of recombinant human plasma RBP4 using the Pichia pastoris expression system. Simple and rapid purification allowed us to obtain 5mg/L of purified protein from the fermentation supernatant with no need to perform denaturing and refolding steps. The identity of the protein was verified by ion-trap MS and Western blotting. The functionality of recombinant RBP4, i.e., the binding to its physiologic ligand, retinol, and the interaction with transthyretin (TTR), was tested by fluorimetric and pull-down assays, respectively. The apparent dissociation constant for retinol to the recombinant protein of 2 x 10(-7)M was consistent with published data for native human protein. The recombinant protein interacted specifically with TTR. These results suggest that expression of recombinant human RBP4 in P. pastoris provides an efficient source of fully functional protein in soluble form for biochemical and biophysical studies.

Soluble expression and characterization of a mouse epididymis-specific protein lipocalin6.

Mouse lipocalin6 (mLcn6) was recently identified to be specifically expressed in the epididymis and speculated to may play a role in sperm maturation. However, further studies were hindered due to the bottleneck to obtain enough recombinant mLcn6 proteins. In this article, GB1 tag was successfully applied to improve the soluble expression of mLcn6. Thermal unfolding experiments demonstrate that GB1 can enhance the structural stability of mLcn6. Fluorescence spectroscopy experiments show that mLcn6 prepared according to our procedure has high affinities to both retinoic acid (K(d)=810nM) and retinol (K(d)=210nM). In conclusion, soluble, stable and active mLcn6 was recombinantly prepared with the help of the GB1 tag, which will facilitate the structural and functional studies of mLcn6.