5. Results
5.2 Bacterial expression
December 15, 2020 k1120217 52/109
December 15, 2020 k1120217 53/109
Figure 12: expression of eYFP-mousePRDM9cst-∆ZnF0 (78kDa) with varying IPTG concentration, medium change and culture size. a fluorescence gel of different expression conditions of Quick WC*. Positive control: MBP-eYFP-mPRDM9cst-ZnF. 8% SDS PAGE gel, exposure 15s. b Coomassie staining of Quick WC* with samples incubated on RT (left) and 95°C (right) before gel electrophoresis. Positive control: MBP-eYFP-mPRDM9cst-ZnF. 8% SDS PAGE gel c Western Blot of 0.2 and 1mM IPTG samples with or without media change (lanes 2-5) Positive control: MBP-eYFP-mPRDM9cst-ZnF.
There is a clear fluorescing band without any degradation products visible at the expected height for a protein of 78kDa in size for all different variants and an ECL signal at the correct height in the Western Blot. For all conditions, the same fluorescence strength and the same signal strength in Western blot can be observed meaning that culture size and media change as well as a variation of IPTG concentration don’t have any impact on the expression result. The Coomassie staining also doesn’t show any difference between the different conditions meaning the total protein amount is also not impacted by these variations.
5.2.2 Impact of cell type: BL21-AI vs Rosetta2(DE3)pLacI
Proteins: eYFP-humanPRDM9A-∆ZnF0, hFcIgG-eYFP-humanPRDM9A-∆ZnF0 and YFP- mPRDM9cst-ZnF (expression #2)
While BL21-AI cells are controlled by the araBAD promoter, the Rosetta2(DE3)pLacI cells are under control of a lacUV5 promoter. The BL21-AI strain allows a tight repression of T7 polymerase and thereby expression of the desired protein by addition of glucose to the growth medium (Invitrogen
December 15, 2020 k1120217 54/109
2010) which is ideal for proteins who could be toxic to the cells which cannot be excluded for PRDM9. The Rosetta2(DE3)pLacI strain is a codon-supplemental strain meaning it encodes tRNAs rare in E.coli to overcome differences in codon frequency between the host strain and the mammalian target gene to be expressed (Merck-Millipore).
To inoculate the growth medium, a single colony derived from a fresh transformation was used for the human Prdm9 constructs in BL21-AI cells whereas for all other constructs a glycerol stock was used. The cells grew for 22h at 37°C to an OD600 of 1.8 to 4.4. For induction, the growth medium was replaced by an induction medium. The expression was performed for 7h with the first 2h at 37°C, the remaining time at room temperature. A quick WC* was prepared (see Figure 13).
Figure 13 a fluorescence gel of Quick WC* of eYFP-mPRDM9cst-ZnF (123kDa; lane 5 & 8), hFcIgG-eYFP-humanPRDM9A-∆ZnF0 (113kDa; lane 6 & 9) and eYFP-humanPRDM9A-∆ZnF0 (87kDa; lane 7 & 10) expression #2.Left in glass plates, right without glass plates. 8% SDS PAGE, 5s exposure. b RT Coomassie staining, 8% SDS gel
The expression of the Fc-tagged human PRDM9 didn’t work at all. Comparing the two expression systems, more fluorescing protein is sticking in the slots for the BL21-AI cells than for the Rosetta cells while more expression product shows a band at the correct height for the Rosetta expression for both the remaining human and mouse constructs. For the Rosetta human expression, an additional band is visible at about double the height of the protein (size 87kDa) which could indicate a dimer.
5.2.3 Temperature variation
Protein: hFcIgG-eYFP-humanPRDM9A-∆ZnF0
December 15, 2020 k1120217 55/109
When temperature is reduced, cell processes are slowed down which results in reduced rates of transcription and translation. The slow-down of cellular processes thereby should result in less degradation products, a correct protein folding and increased solubility.
For this experiment (expression #3), 100 mL of growth medium were inoculated directly with 300 µL of fresh transformation mixture and induction followed at OD600 = 0.5 – 0.7 in fresh induction medium after a very long growth time of 21h at 37°C. BL21-AI and Rosetta cells were inoculated but the Rosetta cells still didn’t show any growth so they were discarded. Expression took place at 16°C, room temperature and 37°C overnight.
Figure 14 a fluorescence gel of eYFP-hFcIgG1-humanPRDM9A-∆ZnF0 (113kDa) in glass plates (expression #3) b fluorescence gel without glass plates 8% SDS gel, 5s exposure c Coomassie staining of eYFP-hFcIgG1-humanPRDM9A
-∆ZnF0 left: RT incubation right: 95°C incubation. 8% SDS gel.
Most desired expression product is visible for the 16°C expression condition which can be observed both in the fluorescence picture and the Coomassie staining, especially for the SN*. The expression at room temperature shows less product compared to 16°C but still enough to be used in further experiments. For both the 16°C and room temperature samples, fluorescing protein is sticking in the gel slots meaning conglomerates have formed. A fluorescing smear is visible as well. The denatured samples show that not the overall protein but especially the desired PRDM9 seems to
December 15, 2020 k1120217 56/109
have accumulated in the 16°C SN* fraction. At the 37°C condition, no fluorescent expression product is visible at all indicating the expression did not work at this temperature.
5.2.4 Expression of hFcIgG1-mPRDM9cst-∆ZnF0 Protein: hFcIgG1- mPRDM9cst-∆ZnF0
The plasmid was freshly transformed to Rosetta cells. A single clone was freshly inoculated and grew for 8h at 37°C until OD600= 0.9. Expression of hFcIgG1- mPRDM9cst-∆ZnF0 (expression #4) took place overnight at room temperature. As there was no eYFP-label, only a Coomassie staining and a Western Blot were performed for protein detection (see Figure 15).
Figure 15 a Coomassie staining of hFcIgG1- mPRDM9cst-∆ZnF0 (76kDa; expression #4). 95°C incubation. 8% SDS gel b Western Blot of hFcIgG1- mPRDM9cst-∆ZnF0 SN*. 5s exposure.
In both SN* and WC*, there is a clear band visible for the expression product in the Coomassie staining. The SN* fraction shows more byproducts while the WC* is very clean. The Western Blot also shows a strong signal already for the SN* fraction at a low sample volume of 1 µL which was not reached by many other extracts with the same volume. Degradation products are visible but still there is a clear band at the correct protein height.
December 15, 2020 k1120217 57/109
5.2.5 Expression of eYFP
100 mL growth medium were inoculated from a Rosetta glycerol stock and grown overnight to a density of OD600=1.6. After the induction, the cells expressed eYFP for 6h at room temperature(expression #5).
Figure 16: Fluorescence gel of eYFP (29kDa). 0.05s exposure. 8% SDS gel.
Compared to eYFP-labeled protein expression, the expression of eYFP itself shows a considerably higher yield (extremely short exposure time needed). This could be due to the decreased protein size of eYFP and probably the toxicity of the recombinant eYFP-labeled proteins to the expression cells.
5.2.6 Summary of protein expression
The biggest effect between the different expressions can be seen by varying the temperature. At 37°C, no expression of the desired protein occurs while at room temperature the recombinant protein is expressed which could be improved further with a lower temperature of 16°C.
Another variation in this thesis is the expression cell type used: BL21-AI vs. Rosetta2(DE3)pLacI.
The Rosetta expression product shows less fragmentized PRDM9.
Culture size does not seem to have an impact on protein expression as well as inducer-concentration and media change.
Reduction of inducer concentration does not show an effect in expression in the agent concentrations varied in these experiments.
December 15, 2020 k1120217 58/109
The media change between bacterial growth and induction does not show any difference in expression yield.