Results

Introduction

Plasmodiophora brassicae, the pathogen responsible for Clubroot disease, poses a major threat to Alberta’s canola crops, endangering the agricultural economy, farmers, producers, and overall food security. As outlined in the Project Description, current detection methods, such as PCR/qPCR, often require external lab testing, making them expensive, time-consuming, and inaccessible to many producers, especially in remote areas. This limits early detection and timely intervention, exacerbating the spread of the disease. The development of a more accessible, protein-based detection system could enhance pathogen monitoring by offering a cost-effective, rapid, and on-site diagnostic tool for farmers, thereby improving disease management and protecting crop yields.

Engineering and Designing Nanobody and FAB

Building on the 2023 Lethbridge Collegiate Teams work, which aimed to develop and validate the components for a protein-based detection system, our project, C.R.O.P.S., advanced that goal by focusing on the design and engineering of a nanobody and fragment antigen-binding (FAB) antibody. These antibodies were conceptualized through binding and molecular dynamics simulations to optimize their interactions with the target protein PbEL04 as a way to effectively detect the clubroot pathogen.

Expression and Purification of Designed PbEL04

PbEL04 expression, purification of the recombinant protein was performed using previously determined optimized conditions. Since our engineered proteins have a 6x histidine tag on them, we utilized nickel affinity chromatography as our first purification technique. This was performed using a 5 mL HisTrap HP column, and more details about this experiment can be found in our protocol Notebook. The resulting chromatogram shows protein eluting with increasing percentages of Buffer B; the elution buffer with a high imidazole concentration, as seen in Figure 1. To confirm that the elution fractions contained PbEL04, a western blot was run, these fractions were pooled and concentrated. The second step of purification involved size exclusion chromatography using Superdex 75 Increase, the chromatogram shows protein eluting off corresponding to their specific sizes. 12% SDS-PAGE was run to confirm the fraction containing PbEL04.

We transformed the construct for pbEL04 into Rosetta (DE3) electrocompetent cells and expressed them in 2 flasks of 50​0 ​mL LB media. The cells were grown until an O.D. of 0.6 and induced with 1mM of Isopropyl β-D-1-thiogalactopyranoside (IPTG), then left to grow overnight. Cultures were spun down then lysed chemically with lysozyme and sodium deoxycholate and mechanically by sonication.

Following this, we purified with Ni2+ Affinity Chromatography on the 5 mL HisTrap HP column and observed protein eluting off the resin at a concentration of ~100mM imidazole based on ultraviolet absorbance​ (Figure 1A)​. We confirmed the presence of the desired protein with a western blot, as seen in Fig. 1​B​, with the first species to elute from the column at running to approximately ~21kDa, which was the theoretical molecular weight of our previously optimized PbEL04 construct.

Figure 1. (A) Chromatogram of the purification of PbEL04 on a HisTrap HP column. Eluted volumes correspond to absorbance readings at 280 nm representing the potential protein being eluted. (B) A Western blot using anti-histidine antibodies was done on a 12%SDS-PAGE gel ran at 200 volts for 1 hour, confirming the fractions containing pbEL04 from the chromatogram (A).

Fractions containing pbEL04 were pooled and concentrated to ~5mM. We performed further purification using size exclusion chromatography, using the Superdex 75 increase 30/100 GL and observed protein eluting from the column at a volume of approximately 11mLs corresponding to a size of ~20kDa​ (Figure 3A)​. We confirmed the presence of protein with an SDS-PAGE gel, as seen in Fig. 2​B​, with the species eluting at the highest peak absorbance running to approximately ~21kDa, which was the theoretical molecular weight of our previously optimized PbEL04 construct.

Figure 2. (A) Size exclusion chromatography (SEC) purification of PbEL04 measured at 280 nm. B. Eluted volumes with potential protein correspond with high absorbance at 280 nm. (B) A 12% SDS-PAGE ran at 200 volts for 1 hour, showing purification fractions selected based on the chromatogram (A).

We were able to express and purify pbEL04.

Transformation of Nanobody and FAB Constructs

The transformation of nanobody and Fab constructs into Rosetta and DH5α cells is a crucial step in biotechnology for recombinant protein expression. The process begins with the introduction of plasmids containing the genetic sequences encoding the Nanobody or FAB using heat shock into competent Rosetta or DH5α cells. Both cell lines were selected for our plasmid transformation as they provide different benefits for our protein production. The FAB and nanobody constructs were successfully transformed into both cell lines.

pET28a plasmids were cloned to contain Nanobody or FAB constructs. The plasmids were transformed into Rosetta and DH5α cells. Rosetta (DE3) electrocompetent cells were chosen as they possess additional plasmids that enhance the expression of proteins containing rare codons, which is often necessary for eukaryotic-derived proteins, like the Nanobody or FAB. They are the cell line that is used in the protein overexpression and purification. In contrast, DH5α cells were selected for their high transformation efficiency and ability to maintain plasmid stability, making them ideal for cloning. DH5α cells were used in any plasmid preparation, as they produced a large amount of plasmids containing the protein coding regions.

Figure 3. LB agar growth plates.Rosetta (DE3) cells transformed with our nanobody(A) and FAB (B) construct. DH5a cells transformed with our nanobody (C) and FAB (D) construct.

The plasmids coding for Nanobody and FAB were separately transformed into Rosetta and DH5α cells.

Optimization of Nanobody Overexpression

To test what condition are best for our nanobody overexpression, we ran an expression screening experiment where we determined what media worked best, what temperature incubation grew the most cells and the concentration of ​isopropyl ß-D-1-thiogalactopyranoside (​IPTG) needed, the OD600 to induce at, and time of incubation. By taking samples of the media contained under each condition, at the time of induction, post 3 hour, post 6 hour and overnight, determination of best expression was done by western blot analysis. Based on the expression results as seen in Figure 3, it was determined what condition would be used for the overexpression and purification of nanobodies.

To determine the optimal condition for the expression of nanobody from plasmid transformed into Rosetta (DE3) electrocompetent cells, ​​an expression screening was performed. To accomplish this, we grew an overnight primary culture in LB media and conditions, as specified in Table 1, were set up, with each media flask of 50 mL receiving 0.1 OD600 of the culture.

Table 1. Test Conditions for Nanobody Overexpression

All secondary cultures were grown at a temperature of 37ºC until they were induced at the O.D. and with the amount of IPTG stated in Table 1. Following this, a 1 OD sample was collected after induction, post 3 hours, post 6hr and overnight, all of which were immediately spun down at 13000 xg for 2 min and supernatant was poured off. These sample pellets were resuspended by vortexing in 1mL of 8M Urea and boiled for 5 minutes. 1 OD samples were collected to visualize the amount of nanobody present in the same amount of cells collected from each condition and at each time of incubation using a western blot.

Figure 4. Western blots using an anti-histidine antibody were done on 12% tris-tricine gels ran at 200 volts for 1 hour showing the amount nanobody present in lysed cells grown under different conditions. 1 OD samples were collected post induction (T0), post 3 hours (T3), post 6 hours (T6) and overnight (TO/N). Blot A contains the 0.6% glucose in LB media, 1.2 OD induced in LB media, and 0.1mM IPTG in LB media trials. Blot B contains Terrific Broth media, 37ºC in LB media, and 18ºC in LB media.

We produced the most amount of nanobody using terrific broth when it was left to grow overnight.

Expression and Purification of Nanobody

Nanobody expression and purification of the recombinant protein was performed using the previously optimised conditions. Since our engineered proteins have a 6x histidine tag on them, we utilised nickel affinity chromatography as our purification technique. This was performed using a 5 mL HisTrap HP column, and more details about this experiment can be found in our protocol Notebook. The resulting chromatogram shows protein eluting with increasing percentages of Buffer B; the elution buffer with a high imidazole concentration, as seen in Figure 4. To confirm that the elution fractions contained nanobodies was tris-tricine gel run. Fractions containing nanobody were pooled and concentrated, then ran on the Superdex 75 30/100 GL size exclusion chromatography. A western blot was performed to determine where nanobodies eluted.

We transformed the construct for Nanobody into Rosetta (DE3) electrocompetent cells and expressed them in 4 flasks of 50​0 ​mL TB media. The cells were grown until an O.D. of 0.6 and induced with 1mM of Isopropyl β-D-1-thiogalactopyranoside (IPTG), then left to grow overnight. Cultures were spun down then lysed chemically with lysozyme and sodium deoxycholate and mechanically by sonication.

Following this, we once again purified with Ni2+ Affinity Chromatography on the 5 mL HisTrap HP column and observed protein eluting off the resin at a concentration of ~100mM imidazole based on ultraviolet absorbance​ (Figure 5A)​. We confirmed the presence of protein with a tris-tricine gel, as seen in Fig. 5B​, with the first fraction to elute from the column having a band at approximately ~18kDa, which was the theoretical molecular weight of our nanobody.

Figure 5. (A) Chromatogram of the purification of nanobody on a HisTrap HP column. Eluted volumes correspond to absorbance readings at 280 nm representing the potential protein being eluted. (B) Tris-Tricine gel confirming the fractions containing proteins in the chromatogram (A).

Fractions containing nanobody were pooled and concentrated to ~5mM. We performed further purification using size exclusion chromatography on Superdex 75 30/100 GL and observed protein eluting from the column between the volumes of approximately 8-26mLs (Figure 6A)​. We ran all peaks from the chromatogram on a western blot with anti-histidine antibodies to confirm which peaks contained the presence of the nanobody, as seen in Fig. 6B​. The peaks at approximately 13mL and 25mL were pooled and concentrated after confirmation they contained nanobody by the appearance of bands.

Figure 6. (A) Size exclusion chromatography (SEC) purification of nanobody measured at 280 nm. Eluted volumes with potential protein correspond with high absorbance at 280 nm. (B) Western blot using anti-histidine antibodies confirming the presence and purification of nanobody in certain fractions selected based on the chromatogram (A).

We were able to confirm the expression and purification of nanobody.

Future Direction: The Test Strip (Lateral Flow Assay)

After confirmation of binding between our recombinant nanobody and pbEL04 have been confirmed we plan to make a lateral flow assay (LFA) that will serve as an easy way to confirm the presence of clubroot on site. LFAs are a commonly used method to determine the presence (or absence) of a molecule of interest, often specific to a disease or condition. For example, COVID19 testing kits utilize the LFA to provide quick and accurate results with a red color change on the strip indicating that you have contracted COVID19. Furthermore, LFA’s are designed in one of two methods: figure 7(A) shows the sandwich format, and figure 7(B) is the competitive format. Both have been used for the detection of various diseases or conditions commercially.

Lateral flow assays provide a qualitative method of detection for specific antigens in solution. If binding is confirmed between pbEL04 and our recombinant antibody via direct ELISA we can then get started on making and testing the LFA. That being said, results from our dry lab showed that our FAB and pbEL04 had unfavorable interactions for binding, making the sandwich format less feasible currently. Furthermore, nanobody and FAB both recognize the same epitope on pbEL04 which is less than ideal for the sandwich LFA as the two will compete for binding on pbEL04. If we are going to use the sandwich format a new epitope region on pbel04 should be utilized and possibly a different antibody scaffold should be used for the immobilized antibody. Therefore, the most feasible first iteration of the LFA would be a competitive format. As It only needs a single antibody (in our case a nanobody) and lab purified pbEL04 as seen in figure 6.

Figure 7. Proposed overview of the parts and mechanism of a lateral flow assay for Clubroot detection via the presence of pbEL04. (A) showing a sandwich LFA where an aliquot containing pbEL04 is delivered to the sample pad, capillary forces then move the sample through the conjugate pad; where our nanobody labeled with colloidal gold binds to pbEL04. As the sample travels over the test line a different immobilized antibody(FAB) binds to the pbEL04 conjugate, causing a red color change. The control line will utilize a rabbit IgG and goat anti-rabbit IgG secondary antibody to ensure that liquid is flowing through the strip. The competitive format (B) uses a single immobilized antibody(nanobody) and lab purified colloidal gold pbEL04. In this format the native pbEL04 in the soil will out compete our synthetic pbEL04 causing no color change on the test line if it is present in the soil.

Another important aspect to optimize the LFA is covalently attaching the labeling nanoparticle (colloidal gold) to either the nanobody or lab purified pbEL04. The reaction scheme for this process can be seen in Figure 8. Moreover, for the reaction to work, colloidal gold has to be carboxylated. Although the scheme presented in figure 8 shows the attachment of colloidal gold to an antibody the exact same mechanism will work if the antibody is replaced with a protein (i.e pbEL04).

Figure 8. The reaction scheme for the covalent attachment of colloidal gold to a recombinant antibody. The reaction is done at high pH (8-9) and the addition of the antibody should occur a few minutes after the addition of EDC and Sulfo-NHS as they form a very reactive and unstable intermediate with carboxylated colloidal gold.