Supported by National Institutes of Health grant R01EY018144.

1. Production, Refolding, and HPLC Purification of Holo-RBP
<ol>
	<li>Transform BL-21cells with the pET3a vector harboring the cDNA for human RBP with 6x His tag on the N-terminus. Grow the transformed BL-21 cells in a shaker at 37 &deg;C in 40 ml LB media with carbenicillin until O.D. at 600 nm reaches 0.5. Induce RBP protein expression by adding IPTG to 1mM. Grow the bacteria at 37 &deg;C another 5 hr.</li>
	<li>RBP produced in E.coli is mostly present in insoluble inclusion bodies. To enrich inclusion bodie...

We share here an optimized RBP production protocol because RBP production and purification procedures are critical to generating correctly folded RBP. Given the possibility of misfolded RBP species and the presence of trace amounts of bacterial proteins even in HPLC purified bacteria-produced RBP, it is helpful to use native RBP from serum to confirm a conclusion related to RBP. Urine RBP, which is commercially available, is a complex mixture of many species of RBP including apo-RBP and holo-RBP 48,49.
<p...

Vitamin A is an organic molecule that is essential for human survival and the proper functioning of almost all human organs. Vitamin A derivatives (retinoids) participate in diverse biochemical and cellular events including the sensing of light for vision 1,2 and the regulation of gene expression and protein translation during embryonic development and in adult tissues 3-6. Although retinol has the ability to diffuse systemically, evolution came up with plasma retinol binding protein, a specific carrier protein for vitamin A transport in the blood to achieve high efficiency and specificity and to avoid toxicity associated with random diffusion 7-10. A high-affinity receptor that binds to RBP and takes up vitamin A was hypothesized in the 1970s 11-13. Despite evidence accumulated in three decades on the existence of the RBP receptor 14-31, the receptor hypothesis was debated for many years due to the existence of an incorrect definition of holo-RBP. The correct definition of holo-RBP is that it is the high affinity 1:1 complex between retinol and RBP. Repeated extraction of holo-RBP by organic solvent is necessary to produce apo-RBP. This definition is used by almost all labs studying RBP 7,9,32-35 or the RBP receptor 14-31,36-42. The incorrect definition of holo-RBP that was used to disprove the existence of the RBP receptor is the acute mixture of free retinol with apo-RBP. Since the function of the RBP receptor in vitamin A uptake from holo-RBP is to release retinol from holo-RBP, the RBP receptor would play no role in retinol uptake if retinol is free to begin with (as proposed by the incorrect definition of holo-RBP).
The recent identification of RBP receptor as a multitransmembrane domain protein called STRA636 and its function in vitamin A uptake from holo-RBP 36-43 strongly argues against the hypothesis that RBP does not need a receptor to deliver vitamin A. Detailed analyses revealed that STRA6 has 9 transmembrane domains with the N-terminus located extracellularly and C-terminus located intracellularly 40. Located between transmembrane 6 and 7 is an essential RBP binding domain 39. STRA6 is coupled to both LRAT and CRBP-I in vitamin A uptake from holo-RBP, but neither LRAT nor CRBP-I is absolutely required for enhanced STRA6 activity 41. STRA6's ability to catalyze retinol release from holo-RBP is the key to its vitamin A uptake activity 41. By relying on STRA6 to release its retinol, vitamin A delivery by RBP can transport vitamin A to target cells in peripheral tissues with high specificity and efficiency.
The critical importance of holo-RBP definition and preparation is illustrated not only by the historical debate on the existence of the RBP receptor, but also by three related recent papers based on holo-RBP definitions different from the original and correct definition 44-46. The first paper used the holo-RBP definition that was used to disapprove the existence of the RBP receptor to study the RBP receptor 44. The second and third papers came up with a third definition of holo-RBP that made it even less likely for retinol to be studied to form a proper complex with RBP 45,46. These studies prepared 3H-retinol/RBP by mixing holo-RBP (not even apo-RBP) with 3H-retinol. Since this assay did not have 3H-retinol/RBP formed and did not remove excessive free 3H-retinol 45,46, it is not an assay for 3H-retinol uptake from 3H-retinol/RBP, but is a free 3H-retinol diffusion assay. It has been shown previously that STRA6 does not enhance cellular uptake of free retinol by LRAT 38 or CRBP-I 41. Virtually all retinol is bound to RBP in the blood and there is no detectable free retinol. A main function of the RBP receptor is to catalyze retinol release from holo-RBP during retinol uptake from holo-RBP 41. If the retinol is artificially released or is in the free form to begin with 45,46, the RBP receptor is not needed. The dramatically different results obtained from the free retinol diffusion assay as compared to assays based on correctly prepared holo-RBP illustrate that correct preparation of RBP is crucial for its functional assays.
RBP can be purified from human serum 41, but the procedure is complex and the yield is low. An alternative approach is to produce RBP in E. coli. Because E. coli does not have the ability to correctly fold mammalian secreted proteins with more than one pair of disulfide bonds like RBP, it is essential to refold RBP and purify the correctly folded protein. Misfolded proteins not only behave differently from corrected folded RBP in various assays, but also cause protein aggregation during storage. For the same reason, apo-RBP is only produced from high-quality holo-RBP. We describe here an optimized protocol to produce high quality RBP 100% loaded with retinol through bacterial expression, refolding, and HPLC purification. HPLC purification not only removes incorrectly folded RBP but also significant bacterial contamination that can cause serious artifacts if RBP is used in signal transduction assays. We also describe two sensitive real-time monitoring techniques to study retinol transport by STRA6. Both techniques depend on high quality RBP. Due to space limitations, the classic techniques of radioactive retinoid-based and HPLC-based vitamin A uptake assays are not described here.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>guanidine hydrochloride</td>
 <td>EMD</td>
 <td>5010</td>
 <td></td>
 </tr>
 <tr>
 <td>cystine</td>
 <td>Sigma</td>
 <td>C8755</td>
 <td></td>
 </tr>
 <tr>
 <td>cysteine</td>
 <td>Sigma</td>
 <td>C7352</td>
 <td></td>
 </tr>
 <tr>
 <td>EDTA</td>
 <td>Fisher</td>
 <td>BP118-500</td>
 <td></td>
 </tr>
 <tr>
 <td>Tris</td>
 <td>Fisher</td>
 <td>7786-1</td>
 <td></td>
 </tr>
 <tr>
 <td>DTT</td>
 <td>EMD</td>
 <td>3860</td>
 <td></td>
 </tr>
 <tr>
 <td>retinol</td>
 <td>Sigma</td>
 <td>R7632</td>
 <td></td>
 </tr>
 <tr>
 <td>carbenicillin</td>
 <td>Fisher</td>
 <td>BP2648-5</td>
 <td></td>
 </tr>
 <tr>
 <td>IPTG</td>
 <td>EMD</td>
 <td>5810</td>
 <td></td>
 </tr>
 <tr>
 <td>PBS</td>
 <td>EMD</td>
 <td>6508</td>
 <td></td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>Fisher</td>
 <td>BP358-10</td>
 <td></td>
 </tr>
 <tr>
 <td>Ni-NTA</td>
 <td>Qiagen</td>
 <td>1018244</td>
 <td></td>
 </tr>
 <tr>
 <td>imidazole</td>
 <td>EMD</td>
 <td>5720</td>
 <td></td>
 </tr>
 <tr>
 <td>heptane</td>
 <td>EMD</td>
 <td>HX0295-1</td>
 <td></td>
 </tr>
 <tr>
 <td>Blocker Casein</td>
 <td>Pierce</td>
 <td>37528</td>
 <td></td>
 </tr>
 <tr>
 <td>Amicon Ultra 15 concentrator (MWCO 10 K)</td>
 <td>Millipore</td>
 <td>UFC901024</td>
 <td></td>
 </tr>
 <tr>
 <td>Microfluor-2 plate </td>
 <td>Fisher</td>
 <td>14-245-177</td>
 <td></td>
 </tr>
 <tr>
 <td>Hamilton syringe Gastight #1710</td>
 <td>Fisher</td>
 <td>14-824-655</td>
 <td></td>
 </tr>
 </tbody>
 </table>

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.

We present here representative results for holo-RBP production and purification by HPLC (Figure 1), real-time analysis of STRA6-catalyzed retinol release from holo-RBP and retinol loading into apo-RBP (Figure 2) and real-time analysis of STRA6-catalyzed retinol transport from holo-RBP to EGFP-CRBP-I (Figure 3).
Without refolding, RBP produced in bacteria is almost completely misfolded due to the presence of many incorrect disulfide bonds. Ther...

Watch this Scientific Journal Video about Real-time Analyses of Retinol Transport by the Membrane Receptor of Plasma Retinol Binding Protein at JoVE.com

Real-time Analyses of Retinol Transport by the Membrane Receptor of Plasma Retinol Binding Protein

Here we describe an optimized technique to produce high-quality vitamin A/RBP complex and two real-time monitoring techniques to study vitamin A transport by STRA6, the RBP receptor.

Vitamin A is essential for vision and the growth/differentiation of almost all human organs.  Plasma retinol binding protein (RBP) is the principle and ...