The overall goal of this procedure is to discover novel extracellular protein interactions involved in cellular recognition processes, in particular membrane embedded receptor proteins. The AveXis approach is designed to circumvent the very weak interaction affinities that typify this class of protein interactions and the fact that membrane proteins are difficult to biochemically manipulate. This is accomplished by first compiling a recombinant protein library of the extracellular regions of cell surface receptors.
This is achieved by designing expression plasmids to produce the ecto domain regions of the receptors as soluble fragments as both monomeric biotinylated baits and pentamer enzyme tagged praise. The second step is to express and normalize the proteins to be ready for interaction screening. All the proteins are produced in mammalian cells to ensure that post-translational modifications such as diss, sulfide, bonds and glycosylation are added.
A crucial step in the protocol is to ensure that the bait and prey proteins within the library are all normalized to within threshold levels. Next comes the exciting part, screening for novel interactions. The Bates are arrayed into individual wells of a microt plate and then probed for interactions using the praise.
Then a wash step is performed. The final step is to add the substrate for the enzyme to detect any captured praise on the arrays of bait proteins. Ultimately, the results of an VEX screen can show which cell surfers receptors are able to form binding partners and may therefore be involved in the recognition and communication processes between cells.
The main advantage of this technique of a popular protein extraction methods, such as the yeast two hybrid or biochemical purifications, is that it is specifically designed to detect extracellular protein protein interactions. It's especially suitable for discovering new interactions between membrane tethered receptor proteins. These interactions are difficult to detect using other techniques because membrane proteins are biochemically difficult to manipulate and typical their counter receptor interactions are extremely weak.
This method can help answer key questions relating to how cells recognize and communicate with each other. At the molecular level, it can be used to answer questions relating to basic biological processes such as which receptor proteins displayed on the surface of eggs and sperm combined each other, or which receptor ligand interactions are responsible for cellular behaviors such as myocyte fusion or neural crest formation. The main challenge with this approach is to compile the bait and pray protein libraries within which to screen for interactions.
It is also very important to make sure that the bait and prey proteins are normalized within the threshold levels required for the assay. This cuts down on the false negative and the false positive frequency. I first had the idea for this method during my PhD when I was trying to identifying a binding partner for the CD 200 cell surface protein.
Essentially, I was asking the question, what is the binding pattern for this particular cell surface receptor? Even today? This remains a technically difficult question to answer at that time, however, the human genome was near in completion and I realized that because we now had a complete list of all of the cell surface receptors in the genome, we could now instead ask within this set of receptor proteins which can interact technically.
This is a much easier question to answer, and AveXis was the method that we developed to answer it. Once a library of bait and pre expression vectors have been established, prepare to transfect them into HEC 2 9 3 E cells begin by seeding the cells the day prior to transfection at a density of 250, 000 cells per milliliter in 50 milliliters of freestyle 2 9 3 media to ensure efficient biotin elation. Supplement the medium used to produce bait proteins, not prey proteins with d biotin with all the plates prepared, incubate the cells overnight in standard conditions.
Because biotin has low solubility and accurate solution, we prefer to weigh out the additional biotin and then add to the media and shake vigorously The following day. Transfect 50 milliliters of cultured cells with 25 micrograms of the bait or prey plasmid using a suitable transfection reagent to produce biotinylated baits cot transfect. The bait construct at a 10 to one ratio with plasmids encoding a secreted form of the ELI protein biotin ligase per a harvest.
The cultures five days post transfection by first paling the cells by centrifugation at 3000 times gravity for 20 minutes and second filtering the S supernatant through a 0.22 micron filter. One of the key features of AveXis is because recombinant proteins are secreted, they can be diluted or concentrated to within threshold activities. We found that recombinant proteins have variable expression up to four orders of magnitude, so the normalization step reduces the occurrence of false positives during the screening steps.
To begin normalization of the bait, unconjugated de biotin must be removed from the media by dialysis since it will compete for the biotinylated bait protein for binding to strept in binding sites begin by securing the bait in a dialysis tube. To ensure effective dialysis of the recombinant proteins, we have found that it is important to provide excess surface area to the dialysis tubing. Typically, we provide two to three times more tubing than is actually required for the volume being dialyzed.
Over a 24 hour period, dialyzed the bait extensively against a suitable buffer such as PBS after dialysis perform. An ELIZA begin by blocking the wells of a strep encoded microt plate with PBST containing BSA for 15 minutes. Meanwhile, on a clean plate, make a series of four one to three dilution of the bait proteins in PBST with 2%BSA.
After the 15 minute incubation, transfer the dilution to the strep TTR encoded plate and incubate the plate for one hour of room temperature after an hour, wash the wells and then to each, well add 100 microliters of two micrograms per milliliter OX 68. Incubate the plate at room temperature for another hour. After the second incubation, do another wash series and load the plate with anti-US alkaline phosphatase.
Incubate the plate for a third hour of room temperature. Now wash the plate three times in PBST and give it a final washing PBS. To remove any residual detergent, add 100 microliters of one milligram per milliliter substrate 1 0 4 in di ethanol amine buffer after an hour.
At room temperature measure absorbance of 4 0 5 nanometers, A dilution series of four different dialyzed bait supinate were detected by Eliza using an anti CD four tag monoclonal antibody. If the undiluted express bait proteins are insufficient to saturate all the biotin binding sites of a strep avid ENC coated plate, then concentrate the protein using a Viva spin concentrator. Otherwise, use the bait protein at a diluted concentration in PBST with 2%BSA.
That is just enough to saturate the biotin binding site of the assay. Quantify the relative levels at which the pre proteins are expressed using the beta lactamase enzyme activity. Begin by making a dilution series of the supinate containing the paraproteins in PBST 2%BSA buffer.
Then add 20 microliters of the dilution to 60 microliters of a nitrous stepin solution loaded in a plate. Immediately transfer the plate to a plate reader and measure the absorbance of 4 85 nanometers once every minute for the next 20 minutes using centrifugal concentrators or dilution PBST with 2%BSA. Set the final concentration of the samples to hydrolyze all the nitros in the assay in about seven minutes, which is about two ol nitros turnover per minute.
Begin the AveXis screen by washing a strep TAVR ENC coated plate in PBST and tapping the plate dry against a paper towel. Next, add PBST with BSA to each well and incubate the plate for 30 minutes. This blocks an spurious protein binding sites.
Within each, well remove the blocking solution with a smart flick of the plate over a sink and add 100 microliters normalized biotinylated monomeric bait to the appropriate wells. Incubate the plate for an hour at room temperature. Then remove the bait samples from the plate with another flick and wash it off three times with PBST.
Then tap dry after the third wash. Now add 100 microliters of the normalized pentamer prey construct to each well and incubate the plate at room temperature for one hour. After an hour, give the plate three more washes with PBST and a final wash with PBS only.
Finally, add 60 microliters of nitrous seven solution to each well and incubate the plate at room temperature for two hours, or preferably incubate the plates for 16 hours at four degrees SIUs. The next day, positive interactions will have turned red. Take a photograph of the plate for a visual record and then quantify the reactions at an absorbance of 4 85 nanometers using a plate reader.
After about 10 minutes from the beginning of the AveXis screening reaction, yellow to red color changes were seen in both the antibody mediated prey capture control and also positive control interaction. This color change indicates hydrolysis of the nitrous in substrate. Some positive interactions are observable on this timescale, but most took a few hours to appear.
Typically, a hit rate of around 0.4 to 0.6%positives has been observed. Once the protein libraries are prepared, the screening part of this technique can be done quickly. It's quite possible to screen up to 12 plates in a day after identifying an interaction.
The most important next step is to firstly show that the interaction can be repeated using fresh independent protein preparations. Secondly, we always determine if the interaction is independent of the bait prey orientation that is, can it be reciprocated? This involves cloning the bait vector domains into the prey vector and vice versa.
Interactions that can be repeated and reciprocated are considered high confidence and have always sh been shown to be positive. Using other techniques such as surface plasma resonance, We like to use surface plasma in resonance to additionally validate any interactions that we re-identify. Importantly, this provides a demonstration that the two purified components can interact directly and can also be used to show that the binding is sat and therefore specific.
