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tRNAs could lead to abortion of protein expression leading to shortened proteins. The Rosetta expression product indeed showed less fragmentized PRDM9.
Culture size did not seem to have an impact on protein expression as well as inducer-concentration and media change.
Nevertheless, aeration usually plays a role in bacterial growth. It is influenced by culture size, shaking speed and the shaking flask shape (baffled shake flasks improve aeration). The culture volume to flask capacity also has to be considered for proper aeration (rule of thumb: not more than 10% of the flask capacity should be filled with culture (Rosano and Ceccarelli 2014)). With optimized higher shaking speed and adjusted culture size, the growth time of the culture, which was extremely long for some expressions, could possibly be reduced. Another factor influencing the growth is the overnight-pre-culture, which was in some cases used to inoculate a larger shake flask.
The advantage of this seed culture is that the cells are growing from lag into log phase already in the small culture allowing inoculation of healthy, well-replicating cells into the large culture.
Additionally, it ensures consistency of inoculated cells, which could also mean a more consistent time for growth until mid-log phase is reached. All in all, this means a more consistent yield of protein expression. The OD600 at the end of growth should not be too high as too many cell divisions have occurred otherwise resulting in "old" bacteria (high cell age), metabolites and many dead cells which could have released toxic substances into the medium which could impact expression afterwards. Nevertheless, expression #2, which took 22h to grow to a very high OD600 of 4.4, showed great results in EMSA experiments. A media change was performed though before expression.
Reduction of inducer concentration usually reduces transcription rate slowing expression down and could thereby improve the solubility and correct folding of the recombinant protein. As no effect in induction agent concentration variation was visible, it may have to be further reduced to see a difference in expression product.
The media change between bacterial growth and induction did not show any difference in expression yield indicating that the nutrients were available in a sufficient amount during growth and expression. Expression outcome could benefit of media change as potentially present metabolites or toxins from dead cells released into the growth medium could be removed. Changing the composition of growth medium could be of advantage. For example, terrific broth (TB) is composed to increase protein solubility and yield compared to the used LB medium (Francis and Page 2010).
Another approach applies osmotic stress by adding sorbitol and betain to prevent the formation of inclusion bodies (Blackwell und Horgan 1991). The amount of correctly folded, active protein could
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possibly be increased by a further media change after expression has finished to a medium containing an antibiotic blocking ribosomal activity or simply without inducer and further incubation for two hours shaking to enable correct folding (Francis and Page 2010).
Furthermore, co-expression of chaperones could be a solution to fold the recombinant proteins correctly.
It has to be said that expressed proteins are less likely to be soluble with increasing molecular weight, especially if they are larger than 60kDa (Canaves et al. 2004). The proteins expressed in this Thesis are in a range between roughly 70 and 120kDa. Fusion tags increasing solubility such as MBP could be advisable but further increase the protein's molecular weight. The same is true for the hFcIgG1-tag, which was used in some expression constructs.
A slow-down in expression considering the given facts is advisable. pH adjustments might improve the results as well.
Lysate preparation
The lysate preparation is an important step to gain soluble, active protein. Different buffers from low to high salt concentration, usage of detergent as well as inclusion body break up were tried in several experiments.
The starting protocol consisted of solution of the recombinant protein by using Sarcosyl as ionic detergent. Most fluorescing protein remained in the WC* fraction indicating the detergent treatment was not sufficient to bring the protein into solution. Therefore, a Freeze-Thaw cycle and sonication steps were introduced to break up inclusion bodies in which the protein could reside. The different buffers used differed significantly in their constitution (see 4.1.1): TBS is a high-salt buffer without any additional detergent or protein-stabilizer, Patel buffer is a medium-salt buffer which contains additional glycerol, ZnCl2 and TCEP to stabilize proteins in solution and TKZN contains significantly less salt (no NaCl) and ZnCl2 and the non ionic detergent NP-40 as stabilizers.
In general, it would have been advisable to always treat part of all samples with the standard protocol for a better comparability as different protein constructs and different expressions under different conditions were performed. Many parameters were changed in between the experiments.
The introduction of Freeze-Thaw cycles and sonication seems to have the greatest impact in protein solubility, probably due to the dissolution of inclusion bodies. Multiple Freeze-Thaw cycles are necessary for efficient lysis. Sonication does not only break up inclusion bodies but additionally
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shears nucleic acid which could be an advantage for following EMSA experiments in which specific DNA-protein interactions should be investigated and bacterial host cell DNA could interfere.
Additionally, it could be possible that some of the protein aggregates are formed due to binding to bacterial DNA, which could be prevented by destroying it with sonication. The use of sarcosyl additionally increased PRDM9 solubility. The amount of detergent needed depends on the CMC (critical micelle concentration), number of aggregates, temperature and nature of the detergent to ensure each protein molecule is isolated in a separate micelle (Thermo Fisher Scientific).
The choice of a nonionic detergent like Triton X100 or Tween 20 or increased NP40 content in TKZN as milder, less denaturing substances than sarcosyl could be of advantage for remaining the protein function.
All used buffer are Tris-buffers, so the temperature is a crucial factor, which has to be kept in mind:
the pH of the buffer is strongly dependent on its temperature. Therefore, it makes a huge difference if the lysis buffer is at room temperature or at 4°C when used which has to be considered at buffer preparation (Tris Base to Tris HCl ratio). In general, the pH could be varied to check if the protein is more soluble and stable at a different pH. It could also be tried to create a buffer with similar conditions like in the cell nucleus where PRDM9 is usually active.
Looking at expression and lysate preparation alone is not enough but the protein's functionality always has to be proven in EMSA binding experiments. In EMSA, the final binding experiments were performed using an extract of expression #2 (YFP- hPRDM9A ΔZnF0, SN*; see Figure 21).
This expression was performed in BL21-AI cells after a fresh transformation and very long growth to a high OD600 of 4.4 before expression took place after a media change at 37°C for 2h and RT for 5h.
Lysate preparation occurred following protocol 3 (sonication, freeze thaw cycles) with 1x TKZN + 0.3% sarcosyl.
Purification
The purification trials using Protein A resin and a hFcIgG1-tag worked, but led to denatured protein.
Therefore, it was no surprise that none of them showed a binding signal in EMSA (see Figure 33).
The proteins were eluated from the Protein A agarose via a pH shift; maybe this treatment was too harsh and resulted in denaturation. The elution conditions could be optimized as well as the dialysis afterwards, which could possibly be replaced by desalting. The time during which the protein is exposed to low pH should be kept as short as possible.
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Additionally, it could be thought of cloning the hFcIgG1-tag or another affinity tag on the C-terminal end of the protein in order to purify only full-length proteins.
Besides affinity chromatography, other purification possibilities could be applied such as ion exchange chromatography (based on charge), gel filtration (based on size), hydrophobic interaction chromatography (based on hydrophobicity) or chromatofocusing (based on isoelectric point).
Purification using the His6-tag failed in previous experiments, maybe due to the usage of a Ni2+
column, which could interfere with the Zn2+ used in the protein's ZnF.
Concentration measurements
Comparison of the Quantitative Western Blot results with the DeNovix measurements showed strong differences and no noticeable proportionality. Western Blot concentrations were higher than DeNovix concentrations as all His-tagged protein is detected in this assay, regardless of the protein's functionality, while absorption measurements can only detect protein with a native YFP label. Additionally, ECL, used for Western Blot signal detection, is a highly sensitive enzymatic reaction, which dynamically changes over time meaning the proportionality between different target signals is constantly changing. In contrast, absorption signal is consistent over time.
In Western Blot, the maximal binding capacity of the membrane could be exceeded at certain areas resulting in an underestimation of strong bands as excess protein is washed away. Proper background subtraction software would also be crucial for more exact analysis of the blots.
Additionally, smaller proteins (<60kDa) are transferred more efficiently onto the membrane than larger ones so the small GFP and the large PRDM9 can probably not be perfectly compared.
Washing and buffer incubation times as well as exposure times play a role in signal detection.
Exposure times should be chosen in a range where strong and faint signals are both visible without saturation.
For both quantification methods, DeNovix and Western Blot, measurement in the linear range is essential. Below the linear range, faint signals are underestimated; above the linear range, strong signals are underestimated (LI-COR Biotechnology GmbH).
DNA binding
Finally, DNA binding experiments were performed with PRDM9. The effects of different binding buffers, incubation times and temperatures of the protein with DNA, membrane types and detection solution manufacturer were investigated.
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The purified protein did not show any binding which was probably due to the elution with a pH shift, which might have been too harsh (see above).
Comparison of different binding buffers showed that a clear shifted band for mouse PRDM9 could be achieved in contrast to Patel300; an effect, which was further enhanced by addition of Sarcosyl.
This ionic detergent might enhance the solubility of the complex, as less fluorescence signal could be detected in the SDS gel slots, where agglomerates were remaining. The more Sarcosyl was added, the less free DNA was visible in an EMSA with human PRDM9, even though no protein-DNA complex was visible. Incubation of the binding worked better at room temperature than at higher temperatures. Incubation time seemed not to have an influence on the binding reaction outcome.
It seems as if the complex has formed and is stable after 1min incubation time at RT already. The more human PRDM9 was added in the binding reaction, the less free DNA could be detected, even though no binding complex was visible. Switching the manufacturer of ECL solution and membrane as well as adjustments in the concentratinos of protein, DNA, Sarcosyl and Poly dIdC showed a shifted band for several human PRDM9 samples. Unfortunately, an unspecific signal deriving from protein without any bound DNA was detected as well so the membrane and ECL might be even too sensitive. After finding EMSA conditions that show a signal for human PRDM9 binding, the 5 fragments of human HS DNA were used for affinity studies using a protein titration. With more protein added, less free DNA could be observed but instead a higher shifted band.
Not all five human HS fragments contain a Myers motif, however, all showed a binding. The binding motif in fragment 4 (without a mismatch) is predicted to be the in vivo binding motif; however, it does not show a stronger binding than the other fragments in the EMSA experiments. Without a negative control of the same size, an unspecific binding could be the reason for this behavior but several groups have also reported PRDM9 binding to DNA not containing an obvious binding motif (Berg et al. 2010; Berg et al. 2011; Pratto et al. 2014; Segurel et al. 2011; Davies et al. 2016; see section 2.3.1). The binding motifs known are not sufficient to predict genome-wide PRDM9 binding, most of them include only contact to a subset of the available ZnF. Altemose et al. have identified DNA binding motifs with variable internal spacing indicating binding of PRDM9 to its target DNA is rather complex than just a simple binding to a consensus motif. These motifs strongly resemble an only recently discovered sparse motif with only 8/22 bases containing substantial information content (Brown und Lunter 2019). The binding specificity of ZnF 1-6 has previously been under-estimated according to Altemose et al. 2017. Depending on its location in the ZnF array, the same individual ZnF can show different sequence specificity (Billings et al. 2013). Striedner et al. showed that PRDM9 binds to target DNA with partially replaced nucleotides indicating that different subsets of
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the complex PRDM9 ZnF domain can confer binding specificity of similar strength (Striedner et al.
2017). Besides specific binding, interaction of hydrogen bonds of the Myers motif's variable positions with amino acids of the ZnF could be shown as well as interactions of the DNA-phosphate backbone with amino acids stabilizing the complex and allowing for some sequence variability adaption (Patel et al. 2016). All in all, binding motifs are not sufficient to predict a PRDM9 binding.
Given all these fact, the binding to each of the five fragments could be explained by PRDM9's binding plasticity. Competition among motifs in close proximity could occur in vivo while in these experiments no competing target site was available in the binding reaction. The affinity of the protein to the HS fragments should be further investigated although it could be different from affinities in the nucleus. In vivo, local chromatin structures probably regulate PRDM9 binding via site accessibility. Thereby, features such as poly-As, epigenetic modifications and chromosomal effects amongst others play a role (Tiemann-Boege et al. 2017). A poly-A microsatellite is in close proximity to the PRDM9 binding motif most likely to be the active one in the hotspot (in fragment 4 of this thesis), which understates this assumption (Heissl et al. 2019).
An EMSA experimental set up could be designed in which hot (biotinylated and therefore detectable) and cold (non-biotinylated) different hotspot fragments are added to the binding assay in order to find out the highest affinity by direct comparison of the different fragments. To verify the binding specificity, a specific cold competitor DNA (same fragment as hot DNA) can be added, which will lead to a weaker detectable signal.
Looking at the EMSA optimization, further improvements would be possible. The EMSA gel run and blot transfer conditions should be kept more constant: due to other experiments performed in the laboratory, the temperature of electrophoresis and transfer could not always be kept constant (freshly prepared buffers at RT instead of 4°C; cooling packs too warm after usage for previous experiments). Concerning the binding reactions, further optimization could be useful to derive a clear shifted band without any smearing, which makes analysis more accurate. The binding buffer is therefore crucial; maybe conditions similar to the nucleus could be tested as this is where in vivo binding of PRDM9 occurs. Poly dIdC as unspecific competitor prevents unspecific binding of other components in the crude protein extract. Usage of proper design of experiments would clearly be of benefit in further optimization as obviously the different factors which were varied are not working independently of each other but variations are in a cause-effect-relationship.
By using a crude protein extract, many other proteins and DNA besides PRDM9 and the specific DNA fragment could have been transferred to the membrane until the full capacity was reached and
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thereby the membrane could have been overloaded resulting in less effective transfer and fixation of specific complex and free fragment DNA. The blocking solution used could be optimized for reduced background, possibly eliminating the unspecific protein signal.
Finally, different exposure times, which make a comparison of the HS fragment experiments difficult, should be consistent. A time series for pictures can be set up at the Biorad imager to find the best exposure time.