The manipulation of cellular function can be achieved through the classical approaches of DNA transfection or viral transduction within the last 15 years. Another method evolved. The protein transduction biological active protein can be directly delivered into mammalian cells using small peptides fused to the protein of interest, which promotes cellular uptake.
As no DNA is used, the risk of exertion agenesis is prevented. In contrast to the above mentioned classical methods. The protein transduction enables delivery of proteins in a titratable manner.
Moreover, small cell populations as well as non dividing cells, such as post mitotic neurons, can be modulated. To express recombinant protein in an efficient manner, we recommend the use of the Paciz vector system. It is a modular vector system consisting of an end terminal and a C terminal variant.
Both vectors allow the easy insertion of your gene of interest for the means of purification. A histidine tag is integrated as well as a protein transduction domain. In our case, touch from the HI virus promoting cellular uptake for cellular distribution.
A nuclear localization signal completes the vector for control reasons fusion tags can simply be removed. Besides the arrangement of those tags can have major influence on the properties of the fusion proteins. The influence on yield and purity as well as sellability and biological function is not predictable.
Hi, my name is Bernard Mus and I'm working in the stem Cell Engineering group in the Institute of Reconstructive Neurobiology in Bond, Germany. Hi, my name is Christoph P and I'm also a member of the in Hoover Lab. Our work group intensively exploits the protein transduction technique to directly deliver biological active protein into various mammalian cells, and today we want to guide you through the process of expression and purification in and from e coli.
Thereafter, we would like to show you how to apply cell permanent fusion protein in cell culture to exemplify the whole procedure expression, purification and application. We make use of the site specific recombination Cree to genetically modify mouse onic stem cells.Okay.Okay. Let's get started.
To inoculate the overnight culture, we make use of already transformed bacteria stored in a glycerol stock at minus 80 degrees centigrade. These bacteria already contain the vector encoding for decre recombination. Using a piper tip, we scratch a small amount of the bacteria onto the piper tip and drop it into the beaker containing the overnight culture.
The overnight culture consists of LB media supplemented with glucose at a final concentration of oh 0.5%and 50 micrograms of carbonic insulin per ml. The beaker is then put into an incubator running at 37 degree centigrade for the expression of the site-specific Recombinate Cree. We are using pre-war TB media for our large scale expression culture.
To speed up the expression, we recommend the use of TB media warmed up to 37 degrees centigrade, which represents the temperature during the expression of the recombinant protein. Next, we add glucose to the TB media at a final concentration of oh 0.5%The glucose prevents an increased basal expression of the fusion protein during the growth of the expression culture before induction. Finally, ampicillin is added to the expression culture at a final concentration of 100 micrograms per ml to assure a pure culture only containing the bacteria which still harbor the plasmid encoding for decree protein.
We now can get our overnight culture and inoculate the expression culture in the ratio of one to 50. The beakers then get transferred into an incubator running at the temperature of 37 centigrade. You should take samples to measure the optical density on a regular basis throughout the expression.
As soon as the culture has reached an optical density of 1.5, the bacteria get induced with a final concentration of oh 0.5 millimolar of IPTG. After one hour of induction, the expression culture is transferred into tubings and subsequently harvested via centrifugation. For this purpose, we are using a SLA 3000 rotor.
The centrifuge is running at a speed of 5, 000 round per minute for 10 minutes at four degrees antegrade. The snat then is discarded and the bacteria pellet can be stored indefinitely at minus 20 degree centigrade. The frozen pellets are resuspended in lizza buffer, a appellate corresponding to one liter of expression culture.
We use 10 mls of lizza buffer wait approximately 15 minutes until the solution gets homogeneous. One milligram of lysozyme is then added per each ML of suspension. The solution is mixed for 20 minutes to allow the enzyme to break open the bacteria.
Subsequently, Benson Nase is added in a dilution of one to 1000 for additional 15 minutes, which will cut the DNA resulting in a non viscous solution. Finally, a son ator is used to ensure lysis of the cells. The program is running for one and a half minutes with a power of 45%after completion of the certification.
Remember to take a sample of every step here, the crude lysate or SDS page analysis of the purification. It is now really important to add one ml of ice cold TSB buffer per ML of suspension as decree protein will tend to precipitate within the glycerol stock. If you're going to skip this step, the solution gets mixed shortly and the crudely is now ready for centrifugation.
For this purpose, we use a SS 34 rotor. The centrifuge is running at 17, 000 RPM for 30 minutes at four degree centigrade. When finished, centrifuge Inc carefully transferred the S supernatant into fresh 50 ml Falcon tubes.
Now add nickel anti A slurry to the supernatant nickel. Anti A is a crucial reagent for the proper purification of the histidine tech fusion proteins. The histidine forms a complex with the nickel ions enabling affinity purification now gently shake the parking tubes for 60 minutes on 40 degree centigrade.
After one hour, you now can apply the suspension onto 20 ML columns and allow the solution to run through by gravity flow. For large scale productions, it is feasible to split the suspension onto several columns. We then wash the column twice with a buffer containing 15 millimolar of innersole.
The amount of buffer applied depends on the resin volume. Two MLS of nickel NTA correspond to one ml of resin for means of washing. We put five resin volumes of washing buffer onto the column after washing, we are now ready to elude our protein.
Therefore, we use an elution buffer with a concentration of 250 millimolar of immuno. So notice how the color of the resin changes towards green. Due to the high concentrations of CRE protein, the OID fraction sometimes gets turd.
To prevent this, mix the solution and add additional lys buffer, the all fractions are now getting pooled and subsequently transferred into a lysis tubing. Lysis will remove the ole first dialyzer step is performed against the buffer containing a high concentration of salt. After one hour, the high salt buffer is exchanged and dialyses procedure is applied overnight.
The next day, the dialyses tubing is taken out of the high salt buffer and transferred into a glycerol buffer. After three to five hours, the glycerol buffer is exchanged and dialyses is again performed overnight. Lysis against glycerol buffer will result in an at least threefold concentrated stock solution.
The stock solution can now be stored at minus 20 degrees centigrade for several years without the loss of activity For quality assurance, you can load your collected SDS page samples onto a gel and stain it subsequently. For Kamasi, the Cree fusion protein is detectable as a prominent band in the OID fraction. In order to use the appropriate concentration of crew recombination, you should determine the concentration via Redford assay.
How to apply so permanent proteins into mammalian cells. First of all, prepare your target cells in order to achieve highest protein transduction efficiencies. See monolayer cells at the density resulting in the 80 to 90%confluence cell layer on the next day in case your target cells are yes cells, which are grown in compact colonies.
Separate the cells and somatically and see them in a single cell suspension five hours in advance. It is important to make a single cell suspension prior to protein transduction because otherwise only air cells on the surface of a colony can be transfused. Aspire the media and watch the air cells briefly with PPS prior to tryin treatment.
Remove remaining Yes, gross media containing serum. After aspirating the PPS overlay cells with dripping and let them incubate three to five minutes. Now check with the microscope if all colony comes are dissolved into single cells.
The reasons just mentioned, this is a crucial step for protein transduction into yes cells. Transfer detached cells into a tube. Place the tube into a table, centrifuge and spin cells down, Aspirate the super agent and resuspend the cell.
P in growth media dilute cells into appropriate cell number. That of course depends on the size of tissue culture. Dish incubate the air cells for additional five hours.
This will give the air cells enough time to attach to the bottom and grow adherent. Three class row stock can be stored at minus 20 degrees for more than two years. When working with recombinant cell permanent fusion protein, it's very beneficial to have such a stock solution that can be used on demand.
While waiting for the cells to attach to the dish, one can prepare the C transduction media simply dilute an appropriate volume of cell permanent creep protein from cholesterol stock into yourself.Media. Since the Clare stock solution is not yet sterile, trans media has to be sterile filtered prior to its application. We recommend using a syringe that can be screwed onto a lobe protein binding filter.
The final creek concentration is dependent on cell type or media supplements. For example, serum has a strong negative influence on the transduction efficiency. This can be overcome by using serum free media or by an increased creek concentration.
We find the optimal condition enabling highest recombination efficiency. Hydrate decree concentration starting from oh 0.5 up to 10 micromolar. We recommend testing different incubation times between six and 18 hours.
After five hours of incubation, take out your target cells and aspirate cross media. Subsequently, we replace it with protein transduction media, put the air cells back into the incubator after overnight incubation. Briefly washed D cells with PPS and changed the protein transduction media to normal ES media.
48 hours after the incubation of cell permanent Cree vent gene expression can be detected via xul. Staining cells with positive bega phenotype have successfully been genetically engineered by the site specific recombination. Cree the report.
The cells harbor CRE use Lae construct upon pre mediad recombination. A lock P flank stop sequence is excised and a luxe reporter gene is activated driven by team promoter. In order to assess recombination efficiency in pre-treated reporter air cells cells have to be fixed.
Aspirate s media wash cells twice with PBS overlay cells with 4%PFA and incubate cells for 10 minutes at room temperature. Thereafter wash cells once again three times with PBS. After aspirating the PBS from the last washing step at Xal Stain solution to the wells incubate cells overnight at 37 degrees in an incubator.
On the next day, Beal activity can be monitored by blue collared cells. Recombination efficiency can be determined by the number of blue colonies. Under optimal conditions, you should be able to achieve a recombination efficiency higher than 90%Alright, today we have shown you how to express and purify site-specific query recombination From e coli.
In this proof of principle study, we were able to induce recombination in most the salts. Okay, that's it. Good luck with your Experiments.I.
