The overall goal of this procedure is to isolate large quantities of functional cystic fibrosis transmembrane conductance regulator, or CFTR protein. This is accomplished first by the heterologous overexpression of the chicken CFTR protein in Charmy visi. The second step is to disrupt the yeast cells and isolate accrued microsomal fraction.
Next CFTR is solubilized and purified. The final step is to measure the rate of a TP hydrolysis by the purified protein in order to assess whether it remains functional. Ultimately, overexpression in s visi allows the production of highly pure, functionally competent CFTR in milligram quantities.
The main advantage of this technique over existing methods, such as expression in mammalian cell lines, is that yeast are easy, rapid, and cost effective to grow in the laboratory. This method can be used to answer key questions in the cystic fibrosis field because the purified protein can be used to study interactions between drugs currently in development and the CFTR protein or even to search for new drugs. This method can provide an insight into CFTR purification and can also be applied to other membrane proteins which are difficult to express and purify.
Generally, individuals new to this method will struggle because CFTR is rapidly degraded by proteolytic enzymes in yeast and has a tendency to aggregate in solution. In this protocol, chicken CFTR will be purified from a previously frozen of S SEI cells. The procedures for growing and harvesting CFTR expressing S SEI are demonstrated in a previous JoVE article.
Microsomes are isolated from the yeast cells as described in the accompanying manuscript, and then resuspended in a minimum volume of CFTR buffer between five and 15 milliliters. A Bradford assay may be used to determine the total amount of microsomal proteins, which should be between 70 and 200 milligrams, and the fluorescence emission spectrum of the membranes should have a distinct GFP fluorescence peak. Two contrasting detergents will be used in the solubilization and purification of C-F-T-R-D-D-M-A non ionic detergent and LPG 14 and ionic detergent.
Prepare the appropriate buffers beforehand following the protocol in the manuscript to solubilize membranes. Dilute the microsomes with an equal volume of the relevant solubilization buffer to give a final detergent concentration of 2%and a microsomal protein concentration of five milligrams per milliliter. Incubate this mixture for one hour at four degrees Celsius with agitation.
After one hour. Centrifuge the mixture at 100, 000 times GE and four degrees Celsius for 45 minutes. Remove and save the supernatant containing the solubilized membrane proteins.
Resuspend the insoluble fraction in 1%SDS solution to a volume equal to the soluble fraction. Measure the fluorescence in this fraction and retain an Eloqua of 50 microliters for SDS page analysis. To begin the procedure for nickel affinity purification of CFTR link to five milliliter nickel Spheros columns in series wash with two column volumes or CV of 20%ethanol, followed by two CV of distilled deionized water After that wash with two CV of solubilization buffer containing one molar MiaSole followed by two CV of solubilization buffer lacking midsole.
Add MiaSole to a final concentration of five millimolar to the solubilized material and load the material into a sample loop of an automated liquid chromatography device after two washes with ole lacking buffer. To remove unbound material, wash the column with three CV of purification buffer with 40 millimolar ole at a flow rate of one milliliter per minute. Collect this fraction in a 50 milliliter tube.
For the second wash, use three CV of purification buffer with 100 millimolar ole. Collect two milliliter fractions in a 96 well plate Elute CFTR from the hiss trap column with three CV of purification buffer with 400 millimolar ole. Collect two milliliter fractions in a 96 well plate monitor fluorescence in eluded fractions retain aliquots of peak fractions.
For SDS page analysis, the remaining peak fraction samples will be used for the next purification step. Begin this procedure by equilibrating the column with 1.2 CV of distilled deionized water, followed by 1.2 CV of gel permeation chromatography or GPC buffer while the column is equilibrating. Concentrate the nickel affinity purified fractions with the highest GFP fluorescence using a 100, 000 kilodalton molecular weight cutoff centrifugal filter at four degrees Celsius.
If purifying in DDM, avoid concentrating the sample above a protein concentration of 0.3 milligrams per milliliter as this will cause significant sample loss. Transfer the RENATE from the concentrators to centrifuge tubes and centrifuge at 100, 000 times G for 30 minutes at four degrees Celsius to pellet large particles. Inject this sample onto the equilibrated column and elute with an is Socratic gradient of 1.2 CV of GPC buffer.
Collect 0.5 milliliter fractions. Measure GFP fluorescence to identify those fractions containing CFTR. Retain a small volume of each for analysis by SDS page to reconstitute CFTR.
Add lipids to the purified CFTR. Add a lipid to protein ratio of 100 to one. Similarly, set up a lipid only control.
Substituting the purified protein with the same volume of GPC buffer. Incubate both samples at four degrees Celsius for one hour. Next, remove detergent from the protein lipid mixture using hydrophobic absorbent beads.
Use 200 milligrams of pre-washed adsorbent beads per milliliter of purified protein and incubate at four degrees Celsius overnight with gentle agitation on the following day, collect the reconstitution sample from the adsorbent beads into a fresh tube using a thin ended pipette tip. C ft R specific APAs activity of the reconstituted CFTR is measured using a modified Chile assay in a 96 well plate format as standards use zero to 20 nanomoles of sodium phosphate diluted in a one-to-one mixture of CFTR buffer and APAs buffer. Incubate both reconstituted CFTR and blank liposomes with one to 100 APAs inhibitors on ice for 10 minutes.
Use at least five micrograms of reconstituted CFTR. Transfer the samples to the 96 well plate and add a TP to a final concentration of two millimolar incubate at 25 degrees Celsius for one hour after one hour. Stop the reaction by adding 40 microliters of 10%SDS to each well.
Next, add 100 microliters of buffer A to each well and incubate for 10 minutes. Then add 100 microliters of buffer B to each well and measure the absorbance at a wavelength of 800 nanometers into a 96 well plate compatible UV vis spectrophotometer. Transpose the absorbance data into a new table.
Convert absorbence at 800 nanometers into an amount of liberated phosphate using the phosphate standards and calculate the rate of a TP hydrolysis following the steps shown. The protocol demonstrated in this video is an efficient means of isolating CFTR enriched microsomes with almost complete recovery of CFTR during the cell breakage and preparation of the crude microsomes. This image of an SDS page gel shows levels of chicken CFTR monitored by Ingel fluorescence of a GFP tag during various centrifugation steps used for microso isolation and washing.
CL indicates cell lysate S indicates supernatants and P indicates pellets. The supernatant after cell breakage and centrifugation at 14, 000 times. G contains virtually all the CFTR including degradation products, ultracentrifugation at 200, 000 times G sediments, all the full length CFTR, leaving some fragments in the supernatant ultracentrifugation at 100, 000 times G of salt washed microsomes pellets.
Nearly all the CFTR with additional removal of some fragments, purification of chicken CFTR in LPG 14 by immobilized metal ion affinity chromatography yielded 80 micrograms of protein per liter of culture at greater than 90%purity. The upper panel shows fractions analyzed by SDS page, followed by kumasi staining. The lower panel shows the same fractions detected by fluorescence of the GFP tag.
The high yield was due to efficient solubilization of CFTR by LPG 14 as indicated by the comparison between lane two loaded with LPG, solubilized microsomes and lane four loaded with insoluble material. In addition, efficient and tight binding to the column resulted in minimal loss of CFTR in the unbound fraction shown in lane three and the absence of CFTR in the wash fractions represented by lanes five and six. The eluded protein had a purity of greater than 90%estimated by kumasi stained SDS page gels and by cytometry of the CFTR and contaminant bands.
Purification of chicken CFTR. In DDM by immobilized metal ion affinity chromatography yielded approximately 50 micrograms of protein per liter of culture of about 60%purity. This figure shows the fractions analyzed by SDS page followed by kumasi staining with fractions prior to elucian on the left and several consecutive EEU fractions on the right.
CFTR is indicated by the arrow later. Fractions are enriched in a 40 kilodalton contaminant, which has been identified by mass spectrometry as ribosomal protein L three. The chicken CFTR purified in either LPG 14 or DDM by immobilized metal ion affinity chromatography was further purified by gel permeation chromatography.
The elucian profile for CFTR purified in buffer containing LPG 14 is depicted by the solid line. While the Ian profile for CFTR purified in buffer containing DDM is depicted by the dashed line, SDS page revealed that LPG purified CFTR indicated by the arrow alluded between eight and 11 milliliters gel permeation chromatography also separated LPG purified CFTR from low molecular weight contaminants. Finally, the APAs activity of the purified proteins was measured.
The results indicate that the purified protein was not able to hydrolyze A TP in the LPG Solubilized state and showed weak APAs activity in the presence of DDM after the addition of lipids and detergent removal. APAs activity was fourfold higher for samples that had been purified in DDM 13 MATP per minute per milligram of protein. The addition of lipids and removal of LPG similarly restored activity to CFTR that had been isolated using LPG, but with the final lower rate than the DDM purified and reconstituted material 1.5 MLEs A TP per minute per milligram protein Once mastered.
This technique can be completed in two days if it is performed properly. While attempting this procedure, it is Important to remember to keep samples on ice at all times following this procedure. Other methods such as thermal stability, electron microscopy, and small angle x-ray scattering can be used to answer additional questions regarding the structure and stability of the protein.
After watching this video, you should have a good idea of how to express and purify large quantities of CFTR protein.
