The overall goal of this procedure is to produce protein complexes by using the multi back baula virus insect cell system. This is accomplished by first generating the multi-gene construct, encoding the protein complex of choice. The second step is to make a composite baula virus that contains the multi-gene construct by transforming bacterial cells and screening for the correct back mids.
Next, isolate the baula viral genome and use it to infect insect cells on a small scale in a six well plate after testing for protein production. The protein complex of choice is expressed on a larger scale and purified. Ultimately, the purified protein complex is used to discover its structure and function and potentially for drug discovery.
The main advantages of this technique over existing echovirus systems are twofold. The MultiPro complexes can be produced with unprecedented. Also, the material produced is of much higher quality because we engineer the echovirus for optimal protein production.
The implication of this technique extend way beyond basic biological questions. Our multi bag platform has been used successfully to produce proteins that are promising new vaccine candidates for a variety of diseases and catalyze cancer research by producing key protein assemblies that cause tumor growth. Generally, individuals new to this method will struggle a bit with the processes and the need for sterility.
So we have worked very hard to establish standard operating procedures to make the application of multi-bank as easy as possible for non-specialist users. Visual demonstration of this method is critical as the robotic steps are much more easily understood when the process is shown in motion. This you cannot extract just by looking at protocols that are written down no matter how detailed.
Prior to starting this procedure, it is important to carefully plan the co-expression strategy. It is recommended that the experiment is first simulated. In silico.
Genes need to be distributed on the acceptor and donor plasmids. What works best is to group together subunits that may form physiological sub modules. Since large complexes have many protein subunits, many genes need to be expressed simultaneously.
The multi back system greatly simplifies the cloning of genes by using automated routines to place genes in small DNA progenitor molecules that contain a short DNA sequence called locks P that is recognized by the Cree recombinase enzyme. These progenitors are then combined in a one step reaction by adding pre recombinase, which concatenates everything that has a lox P site into one large DNA plasmid. This multi-gene construct is subsequently inserted into the multi back bula virus genome.
If a large number of constructs are to be generated, the use of robotic scripts and a liquid handling workstation is recommended. The workstation is equipped with a vacuum for mini preps and PCR cleanup, an EEL for automatic loading of PCR samples. A thermocycler for PCR and incubation steps a shaker for resus suspending cells and cold blocks.
For enzymes, genes of interest are inserted into selected donors and accepters by using restriction enzymes and ligase or alternatively, by using ligation independent methods validate all donor and acceptor constructs, cloned by restriction mapping and sequencing. Proceed diffuse donor acceptor combinations by CRE LAX P recombination to generate the multi-gene expression constructs of choice. The assembled multi-gene constructs are validated by analytical PCR reactions with specifically designed sets of primers.
Snapshots of the robot assisted TR process are shown here. They include the preparation of PCR reactions in multi-well plates, the provision of template DNA and primers, PCR amplification of DNAs and preparation of multi-gene constructs grown in bacterial culture by alkaline lysis in multi-well plates. To begin this procedure, integrate multi-gene transfer vectors into the multi bula viral genome by transforming into DH 10 cells, harboring the viral genome and the functionalities required for TN seven transposition.
After TN seven transposition cells with composite baula virus containing the genes of interest are selected by blue white screening successful TN seven transposition results in a loss of alpha complementation of the beta galacto. Therefore colonies with correct TN seven transposition remain white on selective APLs containing xal. Prepare bacterial cultures from the white colonies and isolate the backin by alkaline lysis and ethanol isopropanol precipitation.
Next transfect insect cells with the purified bao viral genome working in a sterile hood seed aliquots of log phase SF 21 insect cells in a six well tissue culture plate. To each, well add the purified bao viral genome and a transfection reagent mixed in culture media. Incubate the transfected cells at 27 degrees Celsius without motion for 48 to 60 hours.
After 48 to 60 hours, harvest the initial virus or V zero by removing the media replenish with fresh media and return the plate to the incubator two to three days later, test for protein production. And if A YFP marker is present, test for fluorescence for virus amplification. Use V zero the initial virus to infect 25 to 50 milliliters of cells in log phase in small erlenmeyer shaker flasks agitated on an orbital platform.
Shaker count the cells and split the cells every 24 hours until they stop doubling. Follow a low multiplicity of infection or MOI regimen, cells must double at least once, otherwise repeat the experiment with a smaller volume of V zero added. The image on the left shows uninfected insect cells following infection with the multi back baula virus.
Infected insect cells have stopped proliferating and have increased in size as illustrated by the image on the right. After 48 to 60 hours, harvest V one virus by pelleting cells. Remove and store the media containing the virus.
Resus suspend cells with fresh media and transfer them back to the erlenmeyer flask. Remove one times 10 to the six cells every 12 or 24 hours by pelleting and test for protein production and marker protein signal if larger expression volumes are desired. Amplify virus further by infecting up to 400 milliliters of cells in two liter shaker flasks with V one virus.
Following the low MOI regimen, We strongly recommend that V one virus is stored as the production virus by using the ular virus infected insect cells or VIC methods to prevent modifications of the recombinant virus and to preserve high expression levels. 24 hours after proliferation arrest is observed pellet infected cells. At this stage, cells contain complete viral particles just before they would be released into the media.
Remove media resuspend the cell pellet in freezing solution and perform freezing of BIIC in cryo tubes for protein production runs. Use V one V two or frozen BIC aliquots to infect larger cell cultures. Typically 400 milliliters in two liter flasks adhere to the low MOI regimen.
Adjusting the virus volume used for infection such that the infected culture doubles at least once. If A YFP marker protein is present, withdraw one times 10 to the six cells at defined intervals, sonicate and centrifuge the cells and transfer to a 96 well plate measure YFP levels in a standard 96 well plate reader capable of recording fluorescent signals monitor evolution of the YFP signal until a plateau is reached, indicating maximum recombinant protein production. Harvest cells at this stage, store cell pellets at minus 20 degrees Celsius for the short term or minus 80 degrees Celsius for the long term cells should be lyed by a method tailored to the requirements of the protein and then the cytosol and nuclei.
Fractionated nuclear proteins are typically found in the nuclear pellet. While cytosolic proteins remain in the cytosol. Protein purification is greatly simplified by fractionation.
Since a lot of contaminants can be removed in a single centrifugation step. Protein complexes can be conveniently purified from small scale initial cell cultures by utilizing miniaturized purification methods such as multi-well or micro tip based purification. In conjunction with small volume chromatography systems such as the ACTA micro, a future step will be to miniaturize and integrate the entire experimental setup into a small tabletop application that is interfaced with the cloning workstation.
Shown here is the microscale batch robot currently under development, which will be used for this purpose. Strong co-expression of heterologous proteins is achieved by the multi back system as shown here. The overexpressed protein bans are clearly discernible in the whole cell extract, WCE and the cleared lysate.
Sn.The quality and quantity of the protein material produced is often sufficient. Enable structured determination of protein complexes such as the mitotic checkpoint complex MCC shown in this diagram. Often the yield of these analytical purification runs is sufficient for analyzing the structure and function of the purified complex by a variety of means, including electron microscopy.
The standard operating procedures we developed allow non-specialist users to apply our approach with ease and our technology can be used in many laboratories interested in large protein complexes, very much like it's the case today for small protein in Nikola. After watching this video, you should have a good understanding of how to use multi bag in order to produce your protein complexes of interest in unmatched quantity and quality for your ambitious research goals. Working with our systems does not require special safety conditions.
However, keep in mind that the virus will infect you if you are sloppy, even though it'll not replicate in your cells. Therefore, careful handling your steroid conditions is a must while performing this procedure. Our method has become quite popular already more than 600 laboratories worldwide.
Use our technique. We believe that in the future, our method will become an integral part of large scale structural genomics efforts that address the human complex zone. This may help to reveal the basis of diseases and in the long run to lead to new and better drugs and therapies.
