Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

The overall goal of this procedure is to develop a simple, efficient, and scalable method for transient recombinant protein production in a plant-based system using the agro infiltration technique. This is accomplished by first optimizing plant growth conditions. The second step is to transform agrobacterium with launch vectors that combine components of plant viruses and binary plasmids, followed by culturing the transformed agrobacterium for use in the agro experiments.

During agrobacterium infiltration, the plants are flipped upside down and aerial parts are submerged in an agrobacterium suspension. A vacuum is then applied causing air to be evacuated from intercellular spaces in leaf tissues. Through St Rapid re pressurization following release of the vacuum results in the infusion of the agrobacterium suspension into leaves.

Ultimately, immuno blotting and fluorescence assays are used to show recombinant protein production in the infiltrated plants. The main advantage of this technique over existing methods like manual infiltration, is that the vacuum infiltration method can be scaled up for industrial production of recombinant proteins, including vaccines and therapeutic proteins. This method can help answer key questions in the plant biotechnology, and further facilitate the use of plants as an alternative platform for commercial recombinant protein production.

We first had the idea for this method when we found out that scaling up agro filtration in soil growing plants is challenging. For several reasons, soil contains uchin such as viruses, bacteria, nematodes, and in addition, soil content and irrigation water vary from batch to batch and from season to season, and the soil eggs over time in storage. Furthermore, flipping of soil growing plants would result in unwanted dripping of soil into the agrobacterium culture during infiltration.

Demonstrating this procedure will be Rebecca Snow, a senior research assistant from the plant biology and the engineering laboratory, and Emma Gut Robin Lobe. She, she and Adam Trevor, who are research assistant from my laboratory. This study uses hydroponic plant growth medium and nutrient solution to ensure uniformity of plant growth and to eliminate complexities associated with using soil for plant cultivation.

To begin this procedure, soak rock wool slabs in a plant fertilizer solution for five minutes, manually sow seeds on the nutrient soaked rock wool surface. Using the Cedar three Nico Oceana species will be evaluated in this study, two wild type species, Nico Oceana, Benham Ana, and Nico Oceana Excelsior, and a hybrid of Benham, Ana and Excelsior named Nico Oceana. Excela grow plants from the seeds under controlled conditions and a long day photo period in a hydroponic cascading system grow.

Nico Oceana, Bentham Miana, and Nico Oceana Excela for four to five weeks, and Nico Oceana Excelsior for five to six weeks. The Agrobacterium tumor Fasion strains used in this experiment have been transformed with the appropriate launch vectors as described in the accompanying manuscript. For the purposes of this demonstration, five transformed agrobacterium strains are used.

GV 3 1 0 1, carrying a reporter green fluorescent protein, or GFP gene GV 3 1 0 1, carrying the viral gene of the silencing suppressor of tomato bushy stunt virus P 19 and A four GV 3 1 0 1, and a T 10. Carrying the gene of a carrier protein modified liase or lick KM grow agrobacterium strains overnight in LB medium, supplemented with 50 milligrams per liter of can mycin at 28 degrees Celsius with shaking at 200 to 250 RPM on the following day. Prepare agrobacterium suspensions for the desired experiments to examine the feasibility of using multiple agrobacterium strains for transient protein production, dilute a laboratory strain and two wild type strains, harboring the PBI four lick km vector in milli Q water to an optical density at 600 nanometers of 0.5.

To investigate the necessity of inducing agrobacterium virulence genes prior to infiltration first centrifuge agrobacterium cultures carrying the P bid 4G FP vector grown in LB medium at 4, 000 times G for 10 minutes at four degrees Celsius. Then resuspend in MMA induction medium to an optical density at 600 nanometers of 0.5 and stir at room temperature for two hours to test whether acetyl cone enhances transient protein production centrifuge and agrobacterium culture. Carrying the PBI 4G FP vector grown overnight in LB medium and resuspend in infiltration buffer containing one X ms salt 10 millimolar, MES, and 2%glucose.

Divide the cell suspension into four containers. Add a different concentration of aceto cone to each of the agrobacterium suspensions and incubate at room temperature for three hours. To test the effect of co infiltration of a viral silencing suppressor on transient GFP protein production mix.

A milli Q water diluted agrobacterium culture carrying the GFP gene and a culture carrying the viral silencing suppressor P 19 at a four to one ratio During vacuum infiltration of plants, which will be demonstrated. Next is critical to control the vacuum pressure And The duration Of infiltration to vacuum infiltrate plants. First, transfer the prepared agrobacterium suspension to a container inside the vacuum chamber.

Then flip the plant upside down in the diluted agrobacterium and apply a 50 to 400 millibar vacuum for 30 or 60 seconds. The optimal infiltration is routinely applied at 50 to 100 millibar for 60 seconds. Once the vacuum is broken, remove plants from the vacuum chamber, rinse in water, and grow for five to seven days under the same growth conditions used for pre-filtration growth.

To prepare samples for Western analysis, first, collect random leaf samples from Nico Oceana, Benham Miana, Nico Oceana Excelsior, or Nico Oceana Excela plants at four to seven days post infiltration or DPI pulverize the leaf samples in liquid nitrogen to a fine powder. Add three volumes of one XPBS buffer containing 0.5%Triton X 100 to each sample, and transfer the sample to an einor tube. Gently shake or rotate the extracted samples for 15 minutes at four degrees Celsius.

Spin the extract for five minutes and collect total soluble protein into a clean einor tube. Dilute the extracts to an appropriate dilution in one XPBS extraction buffer, and add five x sample buffer to a final one X concentration. Boil samples for five minutes before separating proteins by SDS page.

After proteins have been transferred onto an immobile P transfer membrane and detect GFP using rabbit polyclonal anti GFP antiserum at a dilution of one to 5, 000 in blocking solution. Incubate at room temperature for one hour with gentle rocking after washing the membranes three times with one X-P-B-S-T 20. Incubate with the horse radish peroxidase conjugated anti rabid antibody add a dilution of one to 5, 000 for one hour for GFP detection.

Subsequently, the western blots are processed and band intensities corresponding to the proteins are analyzed as described in the accompanying manuscript. Visual detection of GFP fluorescence in whole transiently transformed plants is accomplished using a handheld long wavelength UV lamp, use a digital camera and an exposure time of 15 seconds to photograph transiently transformed plants through a yellow eight ES 52 filter. Obtain images from western blot analyses using the gene snapped software on a genome.

Quantify the results using the gene tool software with a calibration curve based on a purified GFP standard, Nick Oceana Bentham Miana was grown on Rockwell slabs soaked in commercially available fertilizers to determine the optimal conditions for plant growth and biomass accumulation. The presence of phosphorus is critical to achieve germination and growth of NICO Oceana. Bentham seeds as shown by these six week old plants growing in either a fertilizer solution containing 4.8%phosphorus or a fertilizer solution containing 0%phosphorus.

The effects of agrobacterium growth and infiltration media on plant health and protein production were examined by comparing GFP production in nicotiana. Hamana plants vacuum infiltrated with PBI 4G FP harboring agrobacterium cultures grown in three different media. YEB AB and LB cultures grown overnight in YEB or LB media were centrifuged at low speed and resuspended in MMA induction medium or grown overnight in YEB LB or AB media and directly diluted one to five or one to 10 with Milli Q water as illustrated in this western blot result, plants infiltrated with agrobacterium cultures grown in YEB LB or AB media and diluted with Milli Q.Water showed no significant difference in average GFP production from cultures centrifuged and resuspended in MMA.

Therefore, milli Q water is recommended for diluting agrobacterium cultures for plant infiltration and was routinely used in subsequent experiments to achieve an optical density at 600 nanometers of 0.5. Next, the effects of agrobacterium cell suspension density and time course on target expression were examined. Four different cell suspension densities of agrobacterium carrying PBI 4G FP were assessed and leaf samples were collected at four, seven, and 10 days post infiltration or DPI for western blot analysis at four DPI, there were noticeable differences in GFP fluorescence among plants infiltrated with different cell suspension densities of agrobacterium no GFP expression was observed at a 600 of 0.05 at seven D-P-I-G-F-P.

Fluorescence was similar in plants infiltrated at cell suspension. Densities of a 601.0 0.5 and 0.1, but was lower implants infiltrated at an A 600 of 0.05 in contrast at 10 DPI. No differences in GFP production were observed among plants infiltrated with either of the four cell suspension densities to increase the diversity of agrobacterium strains available for transient protein production.

Nicotiana Benham miana plants were vacuum infiltrated with seven different strains of agro bacteria harboring the vector KBI four lick km. The infiltrated leaves were collected at seven DPI and the level of kinase expression was estimated by Western blot. As shown in this graph, the highest level of liase production was achieved with the strains GV 3 1 10, 1 A four, and LBA 4 4 0 4 with slight differences.

While the lowest level of expression was with C five eight C one intermediate lyase production was achieved with strains a T 77 A T zero six and a T 10 lyase enzymatic activity was demonstrated using a XMO Graham assay. The purified bacterial lyase protein was used as a positive control for enzyme activity. It is interesting to note that nicotiana Benham miana plants infiltrated with the A four and a T seven seven wild type agrobacterium strains showed pathological symptoms such as stunting, pet elongation and curling and leaf curling at seven DPI.

Symptoms were mild in nicotiana benham plants infiltrated with the wild type at T 10 strain. No symptoms were observed in Nico Oceana Benham plants infiltrated with the laboratory strain GV 3 1 0 1. A comparison of plant biomass production in the wild type species revealed that under the same growth conditions, the highest leaf biomass that can be generated from Nico Oceana Excela is approximately twofold higher compared with Nico Oceana.

Benham protein production was examined in Nico Oceana Benham, Nico Oceana Excelsior, and Nico Oceana Excela infiltrated with the agrobacterium strain. GV 3 1 0 1 harboring PBI 4G F-P-G-F-P accumulation was assessed at seven DPI in whole infiltrated leaves. Visual examination of GFP expression under UV light showed an even distribution of GFP in Nico Oceana Hamana and Nico Oceana Excela and an uneven distribution in Nico Oceana Excelsior due to a difficulty of infiltrating an entire leaf area of Nico Oceana Excelsior Western blot analysis of GFP accumulation in these infiltrated leaves at seven DPI showed that the GFP level was higher in Nico Oceana Hamana than in Nico Oceana Excela and Nico Oceana Excelsior the low level of protein production in Nico Oceana.

Excelsior is due to uneven infiltration and uneven distribution of accumulated GFP in the collected leaf. To test the effect of vacuum pressure on the leaves, Nico Oceana Benham plants were infiltrated with the agrobacterium strain, GV 3 1 0 1 harboring PBI 4G FP under vacuum pressures of 400, 200, 100 or 50 millibar at a vacuum holding time of 30 or 60 seconds. No differences in GFP production and mild to no detrimental impact on plant health was observed when vacuum pressures of 5, 100 and 200 millibar were applied for 30 or 60 seconds.

The effect of duration of the vacuum on target expression was assessed by infiltrating one flat of Nico Oceana Benham plants every hour with an A 600 of 0.5 of GV 3 1 0 1 harboring PBI 4G FP for eight hours. In the same agrobacterium culture as shown in this representative result, the level of GFP production was similar at all time, points up to eight hours, suggesting that over this period of time the agrobacterium maintains its ability to launch a single stranded DNA. Since many chemicals and monosaccharides have been reported to enhance transient protein production in various plant species.

This study also assessed the effect of different concentrations of aceto, crinone and glucose. On transient GFP protein production in Nico Oceana Benham, Ana infiltrated with the agrobacterium strain, GV 3 1 0 1 harboring PBI 4G FP.The agro bacteria was grown overnight in YEB media, centrifuged and resuspended to an a 600 of 0.5 either in MMA containing 2%glucose with acetyl serone at the indicated concentrations or in MMA containing 200 micromolar acetophenone with glucose at the indicated concentrations. As shown here, none of the tested concentrations of these compounds induced a significant increase in GFP protein production compared with the control where induction media contained no acetophenone or glucose.

Next, the effect of co infiltration of a silencing suppressor on transient expression of GFP and HAC one genes in Nico Oceana. Benham leaves was examined prior to infiltration agrobacterium GV three one ten one cultures harboring PBI 4G FP and the viral silencing suppressor P 19 of tomato bushy stunt virus were respectively mixed at ratios of one to one, two to one, three to one, and four to one. As indicated by the results of Western blot analysis at seven DPI, the presence of P 19 did not increase or decrease GFP production in nicotiana Benham Miana at any ratio of two of the agrobacterium suspensions.

In addition, the effect of two viral gene silencing suppressors P 23 and P 19 on the prevention of post transcriptional gene silencing for HAC one was investigated. POS of agrobacterium carrying the launch vector PBI four HAC one, and one of the two viral silencing suppressor plasmids were mixed at a ratio of four to one respectively and co infiltrated into Nicotiana Bentham Miana. The infiltrated leaf samples were collected from three to eight DPI.

This graph shows average levels of HAC one expression from three experiments as determined by Western blot analysis. Co infiltration of Nico Oceana Benham with P 23 or P 19 resulted in an increase in HAC one production compared with using no silencing suppressor at 60 pi. This suggests that P 23 and P 19 are efficient in this system.

It was also observed that both in the presence and absence of a silencing suppressor, the level of the HAC one protein production started declining at seven DPI. This indicates that the timing of the decline in the transient protein production in Nico Oceana Benham Miana infiltrated with the launch vector is target specific. Lastly, the stability of the Agrobacterium cell Bank is evaluated every year by infiltrating nicotiana benham plants with the same batch of the Agrobacterium cell bank to evaluate protein accumulation.

These representative results indicate that HA one protein production has been very stable for more than three years. The average HAC one production in Nicotiana benam plants is 651 plus or minus 49.4 milligrams per kilogram. After watching this video, you should have a good understanding of how to apply the agro infiltration technique for production of recombinant proteins at large scale, which can be used for manufacturing subunit vaccine, theo protein antibodies, and diagnostic antigens.

While attempting this procedure, it is important to remember to control the plant cross hydroponically, use viable agro bacteria, control the vacuum pressure and ation, and monitor the vehicle of protein expression. Once master, the agro filtration technique can be done in 24 hour if it is performed properly under controlled conditions.