Recombinant FDH production using Pichia pastoris as host

The complete coding sequence of fdh from B. stabilis 15516 was codon optimized and cloned in the vector pPICZαB in frame of the α-factor secretion signal, inserting a C-terminal Histag for IMAC purification. As mentioned before, expression in P. pastoris and secretion to the medium was preferred as this already serves as a first purification step because P. pastoris secretes little endogenous proteins. Following transformation, three P. pastoris colonies were picked for a first small scale expression test. A sample of the medium was taken, concentrated and analysed by western blot (Figure 5.1). In parallel, cells were lysed and a sample of the lysate was taken along. In the medium, a clear signal was observed for all three colonies at the expected MW of 43 kDa. No signal was seen in the lysate, indicating that the protein production and secretion is efficient and no intracellular degradation occurs.

Figure 5.1: Western blot of lysate and concentrated medium samples after small-scale expression test of three colonies picked after fdh transformation in P. pastoris. FDH (43 kDa) was successfully secreted to the medium.

This secretion appeared to be efficient as no signal was observed in the lysate.

After the small-scale expression test, midscale expression tests were performed, followed by FDH purification from the medium in two steps. Firstly, FDH was purified by IMAC, and secondly, SEC was performed as the polishing step. In a first batch of cultures, methanol concentrations were varied during the expression phase. Standard practice in the lab is the addition of methanol to a final concentration of 1 % for inducing recombinant protein production and additional methanol supplementation every 12 h. Alternatively, the methanol concentration was lowered to a final concentration of 0.5 % for the induction of recombinant protein production and additional supplementation was only done once after 24 h. As methanol metabolism requires a high oxygen supply, oxygen depletion might result in a reduced yield when adding 1 % methanol every 12 h during the expression phase. Lowering the methanol concentration might thus reduce oxygen limitations. Lastly, the methanol concentration was increased to a final concentration of 3 % for the induction of recombinant protein production, and supplemented every 12 h during the expression phase. Higher methanol concentrations have been reported to result in less byproduct formation and to increase recombinant protein production selectivity [388]. The OD600 at the point of collection after 0.5 % methanol addition was 57.3, after 1 % methanol addition 56.6 and after 3 % methanol addition 55.6. Based on these optical densities, the different methanol supplementation strategies did not greatly influence the final cell density. Medium was collected and subsequently purified by IMAC (Figure 5.2). It must be noted that during sample loading after both 1 % and 3 % methanol

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addition, precipitation issues occurred. Looking at the three different chromatograms, using a limited methanol supplementation strategy seemed to result in a slightly higher yield as the 100 mM imidazole elution peak has the highest absorbance. However, together with FDH, contaminating proteins co-eluted with 100 mM imidazole, as seen in the SDS-PAGE analysis of the different elution peaks (Figure 5.3) and these seemed to be relatively more present in case of 0.5 % methanol supplementation. A small amount of FDH eluted at a higher imidazole concentration of 250 mM. The SDS-PAGE analysis of the IMAC elution peaks collected from the medium obtained after 3 % methanol addition did not show any signal and is therefore not shown. In this case, all protein thus appeared to degrade during concentration and buffer exchange. Following IMAC, both 100 mM and 250 mM fractions were combined and further purified by SEC (Figure 5.4). Indeed, no major peaks were seen in case of 3 % methanol addition, confirming that all protein was lost after IMAC (data not shown). In the other two cases (Figure 5.4A and B), several peaks appeared in the chromatogram and all were collected for SDS-PAGE analysis (data not shown). However, no protein was visualized and western blot analysis, being more sensitive, was needed to see which elution peaks were in fact FDH (Figure 5.4C). Purity was therefore not assessed.

Figure 5.2: IMAC purification of FDH after midscale expression tests in P. pastoris. Three different methanol supplementation strategies were tested at 28 °C. A: 0.5 % MeOH every 24 h. B: 1 % MeOH every 12 h. C: 3 % MeOH every 12 h. For all purifications the complete chromatogram is shown on the left. The right chromatogram shows the elution profile in more detail.

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Figure 5.3: SDS-PAGE analysis of IMAC elution peaks, desalted and concentrated after FDH (43 kDa) purification (Figure 5.2). Three different methanol supplementation strategies were tested during the expression phase, i.e.

0.5 % MeOH every 24 h, 1 % MeOH every 12 h and 3 % MeOH every 12 h. The last one is not shown as no bands were seen on the Coomassie-stained gel. The arrow indicates the cut out gel bands, which were identified to be FDH by MALDI-TOF MS.

Figure 5.4: SEC purification of FDH (43 kDa) after midscale expression tests in P. pastoris and a first IMAC purification step (Figure 5.2 and Figure 5.3). Three different methanol supplementation strategies were tested at 28 °C, and only in two cases, FDH was retrieved after IMAC. A: 0.5 % MeOH every 24 h. B: 1 % MeOH every 12 h. C: western blot analysis.

In a next batch of cultures, the temperature during the expression phase was reduced to 16 °C instead of 28 °C, a common strategy to improve the yield of properly folded recombinant protein. By reducing the temperature during the expression phase, the metabolic activity is slowed down, leaving more time for the recombinant protein to fold correctly [339]. Both the 0.5 % and 1% methanol supplementation strategy as described above were tested in parallel. At the point of collection, an OD600 of 54 was measured in case of 0.5 % methanol, and of 50.7 in case of 1 % methanol addition. A minor decrease compared to expression at 28 °C was thus observed. The medium was collected and first purified by IMAC (Figure 5.5). Of note, precipitation issues again occurred in case of 1 % methanol addition. Therefore, the sample loading had to be interrupted, thereby losing the data. For this reason, the chromatogram in

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Figure 5.5B (left) is incomplete. Based on the absorbance of the elution peak at 100 mM imidazole, the yield after expression at 16 °C using 1 % methanol supplementation per 12 h resulted in the highest yield, although many contaminants were observed in this fraction as well.

In both cases, the 250 mM imidazole elution step also contained FDH and the two elution peaks were combined for further SEC purification (Figure 5.7). A smaller peak was seen around an elution volume of 76 ml. Following this smaller elution peak, a broader peak followed with a higher absorption maximum. Based on the subsequent SDS-PAGE analysis (Figure 5.7C), most FDH eluted around 76 ml. The broader peak contained FDH as well, together with lower MW bands and also a smear was observed. Of note, the protein samples were more concentrated than after the 28 °C expression experiments so the gel band intensities cannot be directly compared. It became evident during these experiments that the recombinantly produced and secreted FDH was unstable. Many precipitation issues occurred during IMAC sample loading and smearing was observed on the Coomassie-stained gels after SDS-PAGE.

Figure 5.5: IMAC purification of FDH after midscale expression tests in P. pastoris. Two different methanol supplementation strategies were tested at a lower expression temperature of 16 °C. A: 0.5 % MeOH every 24 h.

The complete chromatogram is shown on the left. The right chromatogram shows the elution profile in more detail.

B: 1 % MeOH every 12 h. The complete chromatogram is shown on the left. Problems occurred during sample loading, leading to loss of data. Therefore the left chromatogram shows misses part of the sample loading data.

The right chromatogram shows the elution profile in more detail.

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Figure 5.6: SDS-PAGE analysis of IMAC elution peaks, desalted and concentrated after FDH (43 kDa) purification (Figure 5.5). Two different methanol supplementation strategies were tested at a lower expression temperature of 16 °C, i.e. 0.5 % MeOH every 24 h and 1 % MeOH. The arrow indicates the cut out gel bands, which were identified to be FDH by MALDI-TOF MS.

Figure 5.7: SEC purification of FDH (43 kDa) after midscale expression tests in P. pastoris and a first IMAC purification step (Figure 5.5 and Figure 5.6). Two different methanol supplementation strategies were tested at a lower expression temperature of 16 °C. A: 0.5 % MeOH every 24 h. B: 1 % MeOH. C: SDS-PAGE analysis.

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In order to address the stability issues, encountered during midscale expression experiments, the stabilizing effect of glycerol was investigated. 5 % glycerol was added to the buffers, used for IMAC, SEC and storage. In a first experiment, the activity was measured after a first IMAC purification. Samples which were purified excluding glycerol and including glycerol in the buffers, were assayed in parallel (Figure 5.8A and B, respectively) after a first freeze-thaw cycle, and retested after a second freeze-thaw cycle. The addition of 5 % glycerol led to an increased stability as the activity was much more retained upon a second freeze-thaw cycle.

Figure 5.8: Activity assay IMAC purified FDH. NADPH formation was measured at 340 nm and the NADPH concentration was calculated with an extinction coefficient ε340 nm = 6.22 x 10³ M⁻¹ cm⁻¹. A: No glycerol was added to the purification and storage buffers. B: Glycerol was added to the buffers. The activity was retained when glycerol was included.

As the activity was retained upon glycerol addition, it was wondered if glycerol addition would lead to less protein loss during purification due to instability and thereby increasing the yield.

In the IMAC chromatogram (Figure 5.9), no big differences in absorbance maxima were seen and subsequent SEC (Figure 5.11A) did not show significant absorbance increase either. The SDS-PAGE analysis after both purification steps (Figure 5.10 and Figure 5.11B, respectively) show that only little FDH was obtained after IMAC and that is was lost after SEC.

Figure 5.9: IMAC purification of FDH after midscale expression tests in P. pastoris, including glycerol in the used buffers. The complete IMAC chromatogram is shown on the left. The right chromatogram shows the elution profile in more detail.

A B

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Figure 5.10: IMAC purification of FDH (43 kDa) after midscale expression tests in P. pastoris, including glycerol in the used buffers. SDS-PAGE analysis of IMAC elution peaks, desalted and concentrated after FDH purification (Figure 5.9)

Figure 5.11: SEC purification of FDH (43 kDa) after midscale expression tests in P. pastoris and a first IMAC purification step (Figure 5.9 and Figure 5.10). A: SEC chromatogram. The numbers indicate the collected peaks.

B: SDS-PAGE analysis of the SEC elution peak after concentration. No protein was recovered.

A B

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