Results
1. Cloning and transformation
Aim
- Insert our gBlock pET-28-His-GR into pET-28a (+) cloning vector
- Transform the cloning strain TOP10 E. coli with our inserted plasmid
- Transform successfully the expression strain BL21(DE3) and identify the desired plasmid
Conclusion
- We successfully inserted our gBlock coding for the Glucocorticoid Receptor Ligand Binding (GR-LBD) with an additional His-Tag using Gibson Assembly Method.
- TOP10 E. coli cloning strain was successfully transformed with our construct plasmid containing our sequence of interest.
We isolated our cloning vector on interest, pET-28a (+) from stock cultures of TOP10 E. coli available in the lab using Qiaprep Miniprep, up-concentrated the resulting linearized vector and used 0.8% agarose gel electrophoresis to confirm the high concentration of the vector in our product.
After confirming the up concentration of our cloning vector of interest, we proceed to clean the PC product with MinElute column and then proceed to assemble our gBlock with the cloning vector through Gibson assembly. TOP10 E. coli cells were transformed with our resulting construct, and single-colony PCR with primers targeting insertion site was performed at the same time as colonies were inoculated in liquid culture.
Insertion of gBlock sequences were successfully determined in gel electrophoresis with the seven bands at ~1000 kb, corresponding to the seven independent colonies of TOP10-pET-28-His-GR. The results of the gel were also confirmed by sanger sequencing of the isolated plasmids.
Figure 1: 0.8% agarose gel showing the band obtained from gel electrophoresis after running for 100 V for 40 minutes. Lane 1 contains the GeneRuler 1 Kb ladder. Lane 2 contains the PCR product of linearization of cloning vector Pet-28a (+).
After this, we proceed with the transformation via Heatshock of BL21(DE3) E. coli cells with our construct TOP10- pET-28-His-GR. Due to time limitations we proceed to start Pre-culture experiments with the transformed cells to confirm the transformation of this cells via induction of the culture using IPTG as the inducer and prednisone as the ligand.
Glycerol stocks of both transformation process were made and kept at -80°C for future experiments, particularly the cryo-stock BL21DE3-pET28-GR.
Figure 2: 0.8% agarose gel showing bands of products from Colony PCR from seven individual colonies (Lane 2 to 8) of transformed TOP10 E. coli colonies with our pET-28-His-GR gBlock. The final lane shows the negative control.
2. Protein expression
Aim
- Confirm successful transformation of our BL21DE3-pET28-GR construct in E. coli BL21(DE3) expression strain.
- Caracterize the small-scale induction of the cultures to obtain the most optimal temperature and time conditions for the incubation of a successfully induced culture to mass-produce our plasmid of interest correctly in a large-scale culture induction.
Conclusion
- We opted to standardize the incubations conditions in 20°C overnight since we obtained more concentrated proteins.
- We confirmed the issues with solubility of our protein of interest reported in the literature while replicating the incubation conditions proposed.
After the transformation of BL21(DE3) E. coli cells we proceed with the small-scale induction of expression to optimize the incubation conditions for our cells. We noticed that most of our protein ended up in the insoluble fraction, as shown in Figure 3. Our protein of interest shows clear bands on the insoluble fraction at ~25KDa with a relatively higher concentration on the condition 20°C overnight conditions.
Figure 4: 4-20% SDS-Page gel image of BL21DE3-pET28-GR expression. The numbers correspond to the specific incubation conditions in Flowthrough fraction (1-5) and insoluble fraction (6-10) following the same incubation periods in each fraction: Uninduced 37°C, 20°C/4hours, 20°C/Overnight, 37°C/4 hours, 37°C/Overnight.
3. Protein purification and future perspectives
Aim
- Test if the large-scale induction of our construct allowed for a most efficient expression of our protein of interest.
- Start the mass-production of our protein by large-scale inducing cultures to be purified in Fast Protein Liquid Chromatography (FPLC).
- Confirm that the protein obtained in the elution fractions of the chromatography corresponds successfully to our protein of interest by performing Western blotting on the regular elution fractions and up-concentrated elution fractions.
Conclusion
- The incubation conditions determined to be optimal in the previous experiments in the small-scale expression showed a successful cultural growth and induction in large-scale expression.
- Successfully confirmed that the protein obtained in the elution fractions obtained in the FPLC corresponds to our protein of interest by performing Western blotting and SDS-Page electrophoresis.
After the transformation of BL21(DE3) E. coli cells we proceed with the small-scale induction of expression to optimize the incubation conditions for our cells. We noticed that most of our protein ended up in the insoluble fraction, as shown in Figure 3. Our protein of interest shows clear bands on the insoluble fraction at ~25KDa with a relatively higher concentration on the condition 20°C overnight conditions.
Figure 5: 4-20% SDS-Page gel image of BL21DE3-pET28-GR protein expression after Nickel-NTA agarose beads with three specific fractions: Flowthrough, First Elution and second Elution.
After the transformation of BL21(DE3) E. coli cells we proceed with the small-scale induction of expression to optimize the incubation conditions for our cells. We noticed that most of our protein ended up in the insoluble fraction, as shown in Figure 3. Our protein of interest shows clear bands on the insoluble fraction at ~25KDa with a relatively higher concentration on the condition 20°C overnight conditions.
We then proceed to start a new cultures while carefully controlling the OD score to obtain a successful colony before adding the inducer and ligand on large-scale cultures. After 2 hours and 50 minutes and an OD score of 0.4235, we proceed to add the inducer and ligand and let it grow under the optimized conditions, 20°C overnight. Mechanical lysis was performed using the French-Press mechanical lysis protocol (Under Protein purification protocol). The filtered supernatant obtained was purified using a HisTag FF column.
The chromatography results of the His-tag purifications shows a specific peak on the elution fraction between T13 to T15 (Figure 6) that likely corresponds to the Eluted protein of interest.
Figure 6: Chromatography of BL21DE3-pET28-GR purification with a HisTrap FF 5 ml column. Conducted with ÄKTA start protein purification system.
We confirmed the results of the chromatography on an SDS-Page Coomassie-stained gel with the main sections of the purification process (Flowthrough, washing step and elution fraction). We confirmed that our protein of interest is being expressed and successfully purified by the bands at ~25kDa molecular weight.
Figure 7: 20% SDS-Page gel image of GR-LBD expression after chromatography purification. The numbers correspond to the Tubes used from the fractions obtained in the chromatography process: 1. Flowthrough (Tube 3), 2. Washing step (Tube 8), 3. Elution fractions (Tube 13 to Tube 20). An additional washing step (4. Tube 10) was added in the final well to improve the visualization of the gel.
At the same time, we started the Western Blot process using the same samples form the purification fractions in an SDS-Page run at 190 v. We incubated the transfer membrane with the MAB050H His-Tag Horseradish Peroxidase-conjugated antibody to target our protein of interest in the fraction parts. As shown in figure 8, it was confirmed that our protein of interest is present in the fractions, however the higher signal detected by the BioRad imager was principally detected in the Flowthrough fractions, with no evident signal in the other fractions.
Figure 8: Western blot visualization of the purified GR-LBD fractions obtained by chromatography.
Even though the western blotting might suggest that we don’t have any concentration of the purified protein in the elution fractions, the SDS-Page gel suggest differently. Therefore, we proceed to up-concentrate the eluted protein Tubes 13 to by precipitations and filtration once again in each separated samples. We compared the concentration obtained with the regular elution fractions by including the original elution samples on the subsequent SDS-Page gels, one for Coomassie staining and the other for western blotting.
Figure 9: SDS-Page Coomassie-stained gel showing the differences in the signal between the up-concentrated Elution fractions and the regular elution fractions of the purified GR-LBD (Tube 13 to Tube 16, for both).
Therefore, we can conclude that we have successfully expressed our protein of interest and demonstrated that the expression and purification experiments that we have done so far with the large-scale induction and FPLC purification. We’re still in our way to improve the purification process and the solubility of our protein of interest, but the results that we have obtained so far are encouraging and a good signal that we’re in the correct path to complete our main goal.
Figure 10: SDS-Page Coomassie-stained gel showing the differences in the signal between the up-concentrated Elution fractions and the regular elution fractions of the purified GR-LBD (Tube 13 to Tube 16, for both).