This protocol shows the production of high quality plasma retinol binding protein that is used to study the activities of retinol binding protein receptor in real time. This is achieved by first expressing and purifying a large quantity of retinol binding protein from bacteria. The purified retinol binding protein is completely denatured and slowly refolded in the presence of its ligand retinol.
Then the correctly refolded retinol binding protein is purified using HPLC to remove incorrectly folded protein and bacterial contaminants. Once high quality retinol binding protein is obtained, it can be used to study the activities of its membrane receptor in catalyzing retinol release, retinol loading, or retinol transport to cellular retinol binding protein one. In real time.
Today I will demonstrate our improved protocol to reform and purify retinal binding protein producing bacteria. This technique can produce high quality retinal binding protein, which is essential for functional assays. I will demonstrate the newly developed techniques to study the receptor for retinal binding protein.
The main advantage of these techniques over existing methods like radioactivity based or HPRC based techniques is that it allows you to monitor the progress of multiple reactions in real time. The most difficult part of this procedure is RVP withholding, which can both determine quality and yield of RVP. If the protein is not produced correctly, the resulting RVP preparation cannot be used for functional acids.
Here we introduce some improvement that we developed over the years that will ensure success. Begin this protocol with the insoluble inclusion bodies from a 40 milliliter culture of BL 21 e coli in which human RBP protein with a six x his tag add 10 milliliters of 7.5 molar guine hydrochloride to the inclusion bodies and sonicate. Then add five milliliters of 25 millimolar triss pH nine and 75 microliters of 10 millimolar DTT, and rotate overnight.
The next day centrifuge at 18 G for 20 minutes at four degrees Celsius to remove the insoluble proteins. After the spin, transfer the supernatant to a new tube and place the tube on ice. Then prepare the refolding buffer on ice as described in the accompanying text.
Next, turn out the room lights to work under a dim red light while vigorously mixing the protein solution in a beaker on ice. Alternately, add 10 microliters of retinol solution for every one milliliter of refolding buffer for a total of 600 microliters of retinol solution and 60 milliliters of refolding buffer. Then continue stirring for five hours in the dark after another centrifugation.
Concentrate the supernatant to about 10 milliliters using Anacon Ultra 15. After concentrating, dilute the sample to 100 milliliters. Using cold PBS.
It is important to remove any precipitate at this point. Then purify the refolded RBP on nickel NTA resin elute in 100 millimolar ole in PBS. Concentrate the EIT using a concentrator with 10 kilodalton molecular weight cutoff to the volume one fifth to one 10th of the original volume.
Then dilute by five to 10 times with tris plus sodium chloride and repeat the concentrating step after concentrating the protein. Transfer it to a 1.5 millimeter micro centrifuge tube immediately before the HPLC run to clear the samples centrifuge at 16, 000 G for 10 minutes at four degrees Celsius. Next, purify hollow RBP on HPLC using an ion exchange column by a sodium chloride step gradient of 220 millimolar for 12 minutes.
360 millimolar for 15 minutes and 1000 millimolar for 15 minutes. Using 25 millimolar triss pH 8.0 as a mobile phase at one milliliter per minute. As the sodium chloride concentration rises, hollow hiss RBP is released.
While APO RBP and misfolded RBP stay bound to the column. If RBP Refolding is not done optimally, misfolded RBP can dominate the whole preparation. Carefully monitor the elution profile of correctly folded and misfolded hiss RBP at 1000 millimolar sodium chloride.
Most of the misfolded hiss RBP should be released. Recover the correctly folded hollow RBP from the peak fractions at 360 millimolar sodium chloride. Following HPLC pool the fractions containing RBP in a 15 milliliter conical tube on ice.
Then use the 10 kilodalton molecular weight cutoff concentrator to concentrate after the volume has been reduced by five to 10 fold. Add PBS to dilute the sample and concentrate again the next day. Use a spectrophotometer to determine the final yield and quality as shown here.
Correctly folded product should have a 330 nanometer to 280 nanometer ratio. Close to one incorrectly folded protein will have a ratio less than one to produce A-O-R-B-P. Add an equal volume of heptane to the purified hollow RBP and mix gently by rotating overnight at four degrees Celsius centrifuge at 16, 000 G for 10 minutes at four degrees Celsius.
After the spin, the top organic phase and the bottom aqueous phase are separated by a white layer. The white layer is denatured APO RBP. To retrieve the aqueous phase, bring the tip of a 500 microliter syringe needle down to the bottom of the tube and carefully aspirate the solution.
Avoid the white material in the interface and transfer the solution to a new tube. Add heptane again and repeat the extraction process three times with a three hour rotation at four degrees Celsius for each after the last extraction, check absorption on a NanoDrop spectrophotometer to make sure the 330 nanometer peak has decreased to the background level. STR RRA six catalyzed.
Retinol release and retinol loading can be monitored in real time by monitoring retinol fluorescence when retinol is bound, there is an increase in fluorescence at 460 nanometers when retinol is released. There is a decrease in fluorescence to perform This assay start by adding 200 microliters of blocker casein to each well of a black 96 well micro Fluor two plate incubate overnight at four degrees Celsius to prevent non-specific sticking of hollow RBP to the plastic. The next day, wash the coated wells of the microflora two plate once with 200 microliters of PBS.
Then after aspirating the PBS cool the plate on ice. Add 100 microliters of PBS to the freshly prepared membranes from one to 5 million cells expressing T RRA six or control cells and pass the membranes through a gas type number 1710 Hamilton syringe six times to produce a uniform suspension. Next, dilute the membrane suspension to one milliliter using PBS.
Add 50 microliters of the membrane suspension to each well and then place the plate back on ice.Here. Realtime monitoring of retinol fluorescence is measured using fluorescence optics of the polar star omega. Set up the instrument as described in the accompanying text with an excitation filter of 320 and the emission filter of 460 dash 10.
Using this instrument perform a retinol release assay in which following a background read hollow RBP is added and readings are taken every 10 minutes for one to three hours. Then perform a retinol loading assay in which all trans retinol is added to the plate. Under dim red light, the plate is read once and then A-O-R-B-P is added.
The reaction is then monitored continuously every five to 10 minutes for one to three hours after all of the measurements have been taken. The raw data will be shown in the Omega data analysis software. Transfer the raw data to Microsoft Excel.
For data analysis retinol EGFP is a fluorescence resonance transfer or fret pair in which retinol serves as the donor and EGFP serves as the acceptor. S stra six catalyzed retinol transport from hollow R BP to EEG FP crbp one is monitored in real time by measuring retinol EEG FP fret prior to performing real-time monitoring of EEG FP crbp one fusion protein with six x hiss tag E GFP crbp one is produced in mammalian cells purified on nickel NTA resin and stored in PBS with protease inhibitors for a short period at four degrees Celsius or at negative 80 degrees Celsius for longer periods. Prepare a blocked microflora two plate and membranes as described in the previous section of this video and add 50 microliters of membrane suspension.
As before. Set the polar star omega with the same settings as before with an additional emission filter of 510 to 10 and simultaneous dual emission fluorescence optics in this assay. EG FP crbp one to one micromolar final concentration is added to each well to start the reaction and a background reading is taken.
Then one millimolar of hollow RBP is added and measurements are taken every five to 10 minutes for one to two hours after all measurements have been taken. Download the raw data as before. Retinol EGFP fret is calculated by the dynamic change in the ratio of the acceptor to donor emission peaks.
The equation to calculate this ratio is shown here where 510 T 510 B, 460 T, and 460 B represent emissions at 510 nanometers after initiation of the reaction at 510 nanometers before a retinol or hollow RBP is added at 460 nanometers after initiation of the reaction added 460 nanometers before retinol or hollow RBP is added respectively. To study the catalytic activities of the RBP receptor STRA six, high quality RBP is produced and purified and then used to monitor stra six catalyzed reactions in real time. This figure shows a dramatic time dependent enhancement of retinol fluorescence in the reactions containing TA six retinol and A-O-R-B-P.
There is little increase in retinol fluorescence in reactions without TA six. This experiment demonstrates stra six catalyzed retinol loading into APO R bp when stra catalyzed retinol transport from hollow R BP to EEG FP crbp one was assessed. There was a time dependent increase in retinol EEG FP fret in the reactions containing TA six.
This suggests that stra six greatly accelerates the transport of retinol from hollow R BP to EEG FP crbp one. When using bacteria produced IBP, it is important to remember that high quality IVP is essential for functional acid involved IBP, and it is important to confirm the result of obtained from bacteria RBP using native RVP from serum. Once high quality RVP is available, it can be used to study the function of RBP receptor in real time using the technique I just described.
These techniques are complementary to the classic, really activity based or HPRC based assays.
