This is a demonstration of a simple method for expression, extraction, and purification of recombinant human IgG fused to GFP in tobacco related Nicotiana benthamiana plants. This protocol can be utilized for purification and visualization of plant produced antibodies, antibody fusions, and most proteins that can be purified through column chromatography. This protocol allows researchers and students at university work labs to visualize GFP tagged proteins at most steps of the protein production process.
Place soil peat pellets on a tray and pour previously boiled, still hot water over the peat pellets for full expansion. Pellets will take a few minutes to expand fully. After pallets are fully expanded, place two to three Nicotiana benthamiana seeds on each peat pellet using tweezers.
After this is done, you'll need to pour about 0.5 inches of water to cover the bottom of the tray. Don't forget to label the tray with the seeding date. Continue to water the seedlings daily with appropriate amounts of fertilizer.
The tray should be covered with a humidome top and place in a growth chamber. The seeded peat pellet should be kept in the growth chamber at 23 to 25 degrees Celsius with a 16 hour photo period and 60%relative humidity. After one week, remove an extra plant from the pellet leaving each peat pallet with only one seedling.
When the plants are two to three weeks old, transfer each peat pellet to an individual pot containing moisture controlled soil. Continue to water seedlings daily with one gram per liter fertilizer, never leave the soil completely dry. Plants are ready for infiltration when they're five to six weeks old.
The following steps must be done next to a Bunsen burner and basic aseptic techniques should be applied to avoid contamination. You will need an Lb kanamycin plate. One microgram per milliliter kanamycin concentration.
strict with a two mutations EHA105 harboring your desired kanamycin resistant construct and grown overnight. EHA105 is one of the most commonly used strains of Agrobacterium two mutations. To begin the culture, fill conical tube with 10 milliliters of lb media, next, at 10 microliters of 100 microgram per milliliter kanamycin, you can also add 10 microliters of 2.5 microgram per milliliter of rifampicin to prevent e-coli contamination.
From the plate, you'll use a single isolated colony verified by PCR to grow your new culture. Choose your isolated colony and inoculate it into the lb. Place the inoculation on the shaker.
incubate at 30 degrees Celsius and 120 to 150 RPM overnight. The next day, if the Agrobacterium culture is drawn to OD600 equaling 0.6 to 0.9 it can be used for infiltration. If it is overgrown OD600 above one one to two milliliters should be transferred to a fresh lb with antibiotics and grown to the required OD600.
Once at appropriate OD600 place the cultures in a centrifuge, making sure that the centrifuge is balanced. Pelt the bacteria by centrifugation at 4, 500G for 20 minutes at room temperature. They can't supernate from both samples.
Then resuspend each pellet and one X infiltration buffer to bring the final OD to 0.4. Combine equal volumes of each IgG fusion construct with the light chain construct, to get the final OD of 0.2 per construct in each tube. Take a paperclip and a five to six week old and benthamiana plant from step one.
Using the paperclip's sharp edge unfolded make a small puncture in the first epidermal layer of the leaf. Avoid puncturing the leaf all the way through. Fill one milliliter syringe without a needle attached with the prepared Agrobacterium solution from step two.
Cover the hole made in the previous step with the end of the syringe. And slowly push to inject the bacteria into the leaf while applying gentle counter pressure from behind the leaf. Try to infiltrate most of the leaf and poke the leaf a maximum of three to four times, excess leaf damage may hinder protein yield.
Watch the leaf darken as the solution is injected without applying too much pressure on the syringe. The infiltrated plant leaf will appear mostly dark from the bottom view. Note that this bacterial solution should be enough for at least three to four plants per construct.
Autoclave any remaining bacterial solution before discarding. Having finished needle-less infiltration, place plants back in growth chamber and continue to water daily. Observed the leaves for chlorosis, necrosis as well as for GFP fluorescence, winter long and short wave UV lamp.
Usually leaves on days four and five show the highest GFP fluorescence. Harvest all the leaves at four to five days post infiltration and weigh the total amount of leaf material. You can use the leaf material immediately for downstream processing, or you can freeze it in negative 80 degrees Celsius until it is ready to use Place the infiltrated plants back in the growth chamber and continue to water daily.
observed the leaves for chlorosis, necrosis, changes in color and leaf tissue death in infiltrated areas. Observed plants for GFP fluorescence. If GFP is present under a long and short wave UV lamp.
Protein expression increases over time, with the highest fluorescence of both GFP constructs generally occurring between days four and five. Harvest all the leaves at four to five days post infiltration and weigh the total leaf material. Use it immediately for downstream processes or freeze at negative 80 degrees Celsius until ready to use.
During the blending process, make sure to keep buffers and blender cups on ice or at four degrees Celsius before use. Place plant tissue from step four into the pretrial blender cup. At chilled extraction buffer containing respective PMSF and sodium ascorbate concentrations to the blender cup.
Place the blender cup on the blender, take a pre-cut sheet of para film and stretch it over the top of the blender cup. Blend the leaf tissue with extraction buffer to imagine 80 with 22nd intervals. You'll have to stir well between blend cycles as needed.
The mixture should appear homogenous without chunks of leaf material. Transfer blended material to a beaker. At a stir bar and stir it for four degrees Celsius for 30 minutes to enhance protein solubility and to allow precipitation of solids.
Place two layers of bare cloth over a clean beaker on ice and pour the extract through it to remove large leaf debris. After all the extract is poured through the mirror cloth fold the mirror cloth to squeeze the residual leaf extract. The extract should appear dark green without visible particulates.
Transfer the extract to centrifuge tubes. Centrifuge the extract at 16, 000Gs for 20 minutes at four degrees Celsius. This will pellet any remaining insoluble material.
Ensure that both tubes are balanced and that the rotor lid is securely tightened. After centrifugation, the pellet should be visible, say supernatant and discard the pellet. Filter the supernatant using a 50 milliliter syringe and glass fiber filter.
Collect 50 microliters of a sample and label crude extract for later analysis. Set up a polypropylene column that holds 20 milliliters of sample. Estimate the amount of slurry needed depending on the target immunoglobulin type and its affinity to the resin.
In this demonstration, we use three milliliters of slurry. Generally three milliliters of total slurry with 1.5 milliliters bed volume is efficient for purification of several milligrams of antibody. Carefully pipette the required amount of resuspended slurry into the capped column.
Open the column outlet from the bottom of the column and allow it to drain until most of the buffer is gone. Immediately pour 10 milliliters of wash buffer one times PBS on top. Let it drain and repeat this wash step two times.
Apply the filtered sample from step five to the column and collect the flow-through. Aliquot 50 microliters of flow-through for later analysis. Save the rest of the flow-through in case the antibody did not bind into the resin.
Wash the resin twice with 10 milliliters of one times PBS to reduce non-specific binding. If desired, aliquot 50 microliters of wash as the buffer drains through the column to verify that the target antibody is not alluded with a wash buffer. While the wash buffer is running through the resin, setup and label five micro centrifuge tubes with 125 microliters of sterile, one molar tris-HCL at pH eight for neutralizing the elution buffer.
Alternatively, add 30 microliters of two molar tris base to get more concentrated eluate. During elution, UV light may be used for visualization. This does not need to be done for the duration of the elution.
If you UV is being used, be sure to wear appropriate personal protective equipment to avoid damage to eyes and skin. A UV light does not need to be used during the elution step. Elute the antibodies by applying five milliliters of elution buffer to the column and collect one milliliter fractions, to each designated tube from the previous step.
Immediately regenerate the column by applying 20 milliliters followed by 10 milliliters of wash buffer. Ensure the resin is not left in an acidic environment for extended period of time. Elution should appear fluorescent.
Often the highest fluorescence is seen in the second elution, but can vary from extraction to extraction. For storage, wash the resin with 10 milliliters of 20%ethanol and PBS and let drain halfway. Recap the top, then the bottom of the column and keep upright at four degrees C.Determine the antibody concentration using a photo spectrometer by measuring absorbance at 280 nanometers.
Store the eluates in negative 80 degrees Celsius and aliquot 50 microliters of each fraction to a separate tube for further analysis. Generally protein durations can be reused up to 10 times without significant loss of efficiency. Refer to the manufacturer's guidelines for specific details determine antibody concentration, using a spectrophotometer by measuring absorbance at 280 nanometers.
Store the eluates in minus 80 degrees Celsius and aliquot 50 microliters of each fraction into a separate tube for further analysis. Analysis of purified protein by Zs page can be done by following standard protocols. Observed fluorescence as a result indicating success of cloning, infiltration and expression of a construct containing GFP.
Over the course of a few days, fluorescents should progressively increase with high fluorescence on days four and five. When protein G is used fluorescent protein binding and subsequent elution from the column indicates purification of an IgG fusion containing as a stable GFP. And the UV exposed gel, you can see only the 25KDa and 75KDa bands of the ladder.
You can also see, the non reduced elution 2 sample. The non reduced elution retains its fluorescence as the relative size one would expect from an intact IgG as its stable GFP fusion. In the coomassie stain gel both reduced and non reduced samples are visualized.
All ladder components are visible, native proteins and IgG fusions can be seen in the total extract supernatant and flow through samples. The wash contains a small amount of IgG fusion. Elution one and four contained less concentrated protein as is expected as most protein has generally eluded from the second and third elution steps.
Elution 2 non reducing can also be seen multiple bands exist in all reducing elution samples. 75KDa indicates a heavy chain fuse to GFP. 50KDa indicates a heavy chain alone, 25KDa indicates a light chain alone, and 10 KDA likely indicates degradation which can be prevented by the addition of protease inhibitors.
This protocol can be used for purification of any antibody fused to any desired target protein. The process can be edited to accommodate various amounts of leaf material, and also allows for the visual determination of protein presence before, during, and after the conclusion of the protein extraction and purification processes. These methods can be useful as controls and can be purpose for teaching techniques.
