The overall aim of this procedure is to express milligram amounts of CFTR protein in Charmy Visier. This is accomplished by first selecting TRANSFORMANT colonies for growth and expression trials in 50 milliliter cell cultures. Next, the cells lies and CFTR expressing colonies are identified.
Then the highest CFTR expressing colonies are grown in a 15 liter fermentor culture. Finally, the cells lies and CFTR containing microsomes are prepared. Ultimately, the results show expression and extraction of GFP fused CFTR through Ingel fluorescence and fluorescence microscopy.
The main advantage of this technique over existing methods for CFTR expression is that it employs a simple eukaryotic host that is cheap, fast, and easy to grow. It has a green fluorescent protein tag that allows quick monitoring of expression levels. And finally, molecular biology steps are minimized in this procedure.
This method can help to answer key questions in the field of cystic fibrosis, such as the biophysical properties of the CFTR protein. Implications of this technique extend towards therapy for cystic fibrosis because it could lead to new drug screens based on the purified protein. New categories of drugs could result from this drugs which interact directly with the protein.
The primary defect in cystic fibrosis should be more specific and potentially have fewer pleiotropic effects. Furthermore, purification of large amounts are the protein are important for obtaining a high resolution structure for the CFTR. Protein structure-based drug design should improve existing drugs and potentially lead to the discovery of new drugs.
Publication of this paper and the accompanying video is linked to the release of DNA or transformed cells through the Cystic Fibrosis Foundation. If you would like to obtain these materials, please contact Elizabeth Joof at the Cystic Fibrosis Foundation. You'll need to complete a materials transfer agreement.
This method can provide insight into the use of A GFP tag for the expression of membrane proteins as introduced by David Drew and his coworkers at Imperial College London. Individuals new to this method may struggle as transformation. Efficiencies can be low and cell harvesting time is critical in order to obtain good expression levels.
We first had the idea for this method during a visit of David Drew and cost us bias to our laboratory. They were undertaking structural studies on membrane proteins that they'd expressed and purified from yeast using this system. Visual demonstration of this method is critical as cell transformation, cell growth after induction and microso isolation are difficult as a publication is linked to the release of a reagent.
The video will also help standardize protocol in order for different labs to compare their results. Demonstrating this procedure will be Tracy Remington and Natasha Kant, who are both graduate students in our laboratory. Liam o Ryan, the other co-author on the paper, is currently in New Zealand and was unable to take part in the demonstration After transforming s visi strain FGY 2 1 7 with a yeast plasmid encoding the C-F-T-R-G-F-P eight hiss gene pick five to 10 well separated colonies from a transformation plate.
Transfer each colony to a separate sterile 50 milliliter Falcon tube containing nine milliliters of yeast nitrogen base or YNB medium and one milliliter of glucose.Medium. Grow the cultures overnight for 16 hours at 250 RPM and 30 degrees Celsius in an orbital shaking incubator to make glycerol stocks for each of the screened colonies. Aseptically add 0.8 milliliters of the overnight cultures to 0.2 milliliters of sterile glycerol in labeled screw top vials.
Vortex briefly and store at minus 80 degrees Celsius dilute the remaining overnight cultures to a final volume of 50 milliliters in YMB with 250 microliters of glucose medium grow the cultures and labeled 250 milliliter erlenmeyer baffled flasks to an OD 600 of 0.7 to 0.8 at 250 RPM and 30 degrees Celsius in an orbital shaking incubator. Induce the cultures by adding five milliliters of galactose medium to each flask. Reduce the temperature of the incubator to 25 degrees Celsius and grow overnight for 16 hours.
Transfer the cultures into 50 milliliter Falcon tubes. Harvest the cells by centrifugation at 3, 500 times G at four degrees Celsius for 10 minutes in a benchtop centrifuge while the centrifuge is running. Prepare two milliliters screw top micro fuge tubes containing approximately 300 microliters of acid washed glass beads and place on ice.
Discard the supernatants and reus resuspend each pellet in 800 microliters of ice cold cell Resus suspension buffer or CRB with protease inhibitors. Transfer the suspensions to the micro refuse tubes and keep them on ice. Delay the cells load into a mini bead beater and run for three minutes.
Place the tubes into a benchtop micro fuge and centrifuge at 3, 500 times G at four degrees Celsius for five minutes. Transfer the supernatants containing the membranes to clean micro fuge tubes and place on ice. Collect the crude membranes by centrifuging at maximum speed at four degrees Celsius in a benchtop micro fuge for two hours.
Discard the supernatant and resend each pellet in 50 microliters of ice. Called CFTR buffer in clean micro refuse tubes. Mix 15 microliters of each suspension with 15 microliters of two times loading dye by pipetting.
Do not boil the samples as this will cause CFTR and other membrane proteins to form SDS insoluble aggregates and also denature the GFP tag. Load the samples as well as a prestained protein standard onto a four to 20%drisk glycine gradient gel, and run it at 150 volts for 40 minutes. After performing ingel fluorescence to identify the highest expressing cells and verifying expression by kumasi staining streak out the highest expressing cell line from its glycerol stock onto a fresh YNBA plate and incubate at 30 degrees Celsius for two to three days.
Store the plate for up to two weeks at four degrees Celsius to prepare pre cultures for the fermentor. Use a sterile loop to scrape a one centimeter square area of cells from A-Y-N-B-A plate and add to 45 milliliters of YMB and five milliliters of glucose medium for an OD 600 of around 0.1. Grow the cultures at 250 RPM and 30 degrees Celsius in an orbital shaking incubator until the OD 600 reaches one.
Split the culture between two two liter erlenmeyer baffled flasks each containing 450 milliliters of YMB medium and 25 milliliters of glucose. Medium grow overnight at 250 RPM at 30 degrees Celsius in an orbital shaking incubator until the OD 600 reaches 1.2. To set up the fermentor, make 11.2 liters of YNB and add an additional 8.28 grams of YNB and 0.95 grams of dropout supplementation to compensate for the addition of glycerol at induction.
Next aseptically, add the 11.2 liters of YNB and 75 milliliters of glucose medium to a sterile 20 liter fermentor vessel and adjust the running temperature to 30 degrees Celsius. Then aseptically, add the pre cultures to the fermentor. Set the stirring speed to around 800 RPM and adjust the compressed airflow to around 15 decimeter cubed per minute.
When the culture reaches an OD 600 of 1.2 induce by aseptically adding 1.5 liters of galactose medium and 1.2 liters of glycerol. Reduce the temperature to 25 degrees Celsius and allow the culture to grow for 16 hours using a peristaltic pump. Transfer the culture into chilled one liter centrifuge tubes on ice.
Harvest the cells by centrifugation for 30 minutes at 3, 500 times G at four degrees Celsius. Resus suspend the cells in 150 milliliters of ice cold micro zone Resus suspension buffer or MRB with protease inhibitors and one millimolar DTT From this point forward carry out all work at four degrees Celsius. Use a cell disruptor to lies the cells.
Then transfer the lysates to 50 milliliter falcon tubes and pellet the cell debris by centrifugation at 3, 500 times G and four degrees Celsius for 15 minutes. To remove mitochondria, transfer the supinate to chilled centrifuge tubes, and spin at 14, 000 times G at four degrees Celsius for 30 minutes. Next to collect microsomes, transfer the SUP natin to chilled ultracentrifuge tubes centrifuge at 200, 000 times G at four degrees Celsius for 90 minutes.
Carefully decant and discard the supine natin and add two milliliters of ice cold CFTR buffer with protease inhibitors and one millimolar DTT to each tube. Using a paintbrush, gently re suspend the pellets. Then top up each tube with CFTL buffer and vortex Centrifuge the suspension at 100, 000 times G at four degrees Celsius for 60 minutes.
Discard the supine agent and use a paintbrush to repeat the Resus suspension process in two milliliters of ice cold CFTR buffer with protease inhibitors and one millimolar DTT. Pull the resuspended microsomes, then adjust the final volume to 50 milliliters with CFTR buffer and mix well. Reserve a one milliliter aliquot for SDS page and store the microsomes at minus 80 degrees Celsius until needed to extract CFTR from the microsomes.
Refer to the written protocol. Transformation of yeast with the CFTR containing plasmid is not 100%efficient. A representative small scale screen of CFTR expression in selected colonies from a transformation experiment will yield about one in four colonies expressing the protein shown.
Here are results from a screen of five colonies picked from a plate. One colony shows a strong level of expression of the C-F-T-R-G-F-P fusion protein. The C-F-T-R-G-F-P fluorescence levels will vary considerably between experiments with colony four showing at least 10 times greater fluorescence than the intrinsic fluorescent band at about 70 kilodaltons.
Even with a high expression level of C-F-T-R-G-F-P as shown on the left, it is unlikely that the C-F-T-R-G-F-P band will be discernible in the cell extract by kumasi staining as seen on the right. This figure shows the presence of C-F-T-R-G-F-P within microsomes. The results of this experiment are important not only to assess the efficiency of the induction of expression, but also to check that the microsomes have been prepared carefully and that proteolysis has been minimized.
As shown here. Growth of cells beyond 16 hours will give rise to decrease CFTR expression. This is likely due to turnover of the protein, perhaps from upregulation of the yeast protein quality control machinery.
It is therefore advisable to monitor CFTR expression levels after induction, using ingel fluorescence and fluorescent imaging While attempting this procedure, it is important to monitor cell growth using OD 600 measurements. Try everything on a small scale first before scaling up and check protein expression as a function of time. After induction, as the optimal time to harvest cells may vary from lab to lab.
After watching this video, you should have a good understanding of how to express milligram quantities of the CFTR protein in the yeast Scro Visier. Following This procedure, the CFTR protein can be purified from the yeast microsomes for further biochemical and biophysical characterization. After its development by David Drew and his coworkers, this technique paved the way for better expression of eukaryotic membrane proteins.
We hope that this work will have an impact on cystic fibrosis research by allowing labs worldwide to produce CFTR using a cheap and easy expression system. This work would not have been possible without the support of the Cystic Fibrosis Foundation and of course, our colleagues on the CFTR three dimensional Structure Consortium. Don't forget that working with any recombinant organism is tightly regulated and handling and disposal Procedures should be approved by the appropriate regulatory body before any work is begun.
