Team:Uppsala/Proof Of Concept
The plan
One of the main goals of our project is to produce our enhanced versions of FGF2 on an industrial scale. In order to prove that this could be achieved, we visited a bio-prototyping facility in Uppsala called Testa Center.
Upscaling at Testa Center
The purpose of the performing experiments at Testa Center was to see if the expression of the FGF2 variants could be upscaled. This is an important factor for reducing the cost of production and making it more accessible for use in cultivated meat. The two strains which had the most promising results (which were based on SDS-PAGE results from a small scale expression at the university lab) were the wild-type FGF2 (FGF wt) and the hyperstable FGF (FGFhs). They were therefore chosen to be expressed at the large bio-prototyping facility. The E. coli strain BL21, containing the FGF2 wt or the FGFhs plasmid were grown in 5 L bioreactors (Figure 1). More information is available at Protocols and also at Notebook.
Figure 1. A 5 L bioreactor with a control unit at Testa Center used for the growth of E. coli strains expressing variants of FGF.
The Optical density (OD) was measured at wavelength 600λ, after the cells reached OD2.0 they were induced with IPTG. OD was measured at 30 min intervals for the first couple of hours to follow the growth of the bacteria and then left to grow in the bioreactor overnight (Figure 2 & 3). The following day the cells were then spun down in a centrifuge set to 4 degrees. The pellet was collected, which had the appearance of a brown paste (Figure 4). This paste was almost entirely E. coli cells which contained FGF2.
Figure 2. Growth of E. coli expression of FGF2 wt in 5L bioreactors.
Figure 3. Growth of E. coli expression of FGF2hs in 5 L bioreactors.
Figure 4. Pellet collected from centrifugation of induced E. coli cells grown in a 5 L bioreactor.
Unfortunately an SDS-PAGE gel revealed that the strain expressing FGFhs had not produced a protein of a similar size to the desired protein size. However, the results for FGF2 wt did show bands of the right size on the SDS gel. Therefore it was decided that only FGF2 wt would be purified using immobilized metal affinity chromatography (IMAC). The culture of the strain expressing FGF2 wt was dissolved in a lysis buffer and lysed in a french press machine. The sample was then spun down and the pellet was thrown away. The FGF2 protein was in the supernatant of the solution as its solubility is increased by the thioredoxin protein. The sample was then applied to the IMAC column (Figure 5), the sample bound strongly to the IMAC column as it was designed with a 6 residue histidine tag which should bind to the nickel ions in the column.
Figure 5. The IMAC column at Testa Center used for purification of proteins expressed from the E. coli strains expressing FGF in the bioreactor.
A concentration gradient of imidazole was used to elute any proteins which had bound strongly to the column. At a high concentration of imidazole, a protein was eluted indicating that a protein had bound strongly to the IMAC column, see Figure 6. This was done by measuring the absorbance of the sample over time, where the peaks in the measured absorbance indicated the protein was eluted. The y-axis shows absorbance and the x-axis shows the amount of liquid applied to the IMAC column. The graph shows the elution of protein at a high concentration of imidazole at roughly 600 ml on the x-axis.
Figure 6. Chromatogram for the elution of FGF2 from the IMAC column. Absorbance (y-axis) measured over the amount of eluent (x-axis) applied to the IMAC column. The absorbance measurement shows the elution of a protein at approx. 600 ml of added eluent.
The samples which correspond to peaks on the graph were run on an SDS-PAGE gel to check if the eluted protein was of the same size as our target protein and this was confirmed as seen in the gels below (Figure 7).
Figure 7. SDS-PAGE of eluted protein from the IMAC column. FT: Flowthrough, W1-2: Wash fractions, B1-B5 & C1: Elution fractions. Elution B5 shows the strongest band of the correct size (~30.8 kDa).
The fractions containing FGF2wt were spin concentrated (Amicon spin concentrators) and the imidazole washed away with a reaction buffer. The protein concentration was measured using a Bradford Assay from Bio-Rad [1]. A standard curve was created using BSA. OD595 for the sample was measured and the standard curve for the BSA was used to calculate the amount of expressed protein (Figure 8). The total protein concentration was 147 mg/mL in 11 mL buffer and the total protein yield from large scale production was 1.6 g. This is an approximately 461 times higher total yield compared to small scale production. The volume of the culture in the bioreactor was 50 times larger than for the small scale expression. This gives a ~9 times higher yield per volume for large scale compared to small scale. However, due to the impurity of the sample (see the multiple bands in Figure 7) it is impossible to say how much of the protein from the large scale purification is FGF2 and how much is protein contamination. The expression in small scale was also done with cells from a different company which might affect the comparison.
Figure 8. Bradford Assay BSA Standard Linear Regression calibration curve used as standard curve for determination of protein concentration.
Summary
Through our upscaling experiments we have cultivated E. coli and induced them to express the FGF2wt protein in bioreactors containing 50 times the volume of our small scale E-flasks. In succeeding in producing large protein yields we have given strong evidence of our FGF2wt construct and expression system being adaptable for large scale production. This shows its potential in the cellular agriculture field, whose ultimate goal is to cultivate meat in quantities large enough to feed the world's growing population in an environmentally sustainable way. We further hypothesize that since we succeeded in expressing the FGF2wt gene in this construct which we have designed, with the expression system we chose, that all our FGF2 variants (which we expressed and purified on a small scale) are suitable for large scale expression.
References
[1] M. M. Bradford, “A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding,” Anal. Biochem., vol. 72, pp. 248–254, May 1976, doi: 10.1006/abio.1976.9999.
