Part:BBa_K5091006:Experience

Engineering and Designing Nanobody

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. This nanobody was conceptualized through binding and molecular dynamics simulations to optimize the interactions with the target protein PbEL04 as a way to effectively detect the clubroot pathogen.

Transformation of Nanobody Construct

The transformation of nanobody construct 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 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 nanobody construct was successfully transformed into both cell lines.

pET28a plasmids were cloned to contain Nanobody. 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. 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.

The plasmids coding for Nanobody was 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 \_, 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.

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.

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 5. 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.

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.

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

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