5. RESULTS AND DISCUSSION
5.1 Production of EPO and incorporation of fluorinated amino acids
5.1.1 Expression and purification of WT-EPO in Escherichia coli
Expression of WT-EPO was performed in E.coli BL21ΔP. This BL21(DE3) derivative was established by Martin Hamann and is an proline-auxotrophic strain.
During genetic modification, the gene for the T7 DNA polymerase was damaged.
Therefore, pET11a-WT-EPO + T7 Pol was used for the expression of WT-EPO. The expression was induced with 1 mM IPTG. After incubation overnight, expression was analysed via SDS-PAGE analysis (Figure 15).
Results and Discussion
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As WT-EPO has two structural disulfide bonds and a quite hydrophobic surface, the protein is expressed in inclusion bodies. Thus, EPO has to be extracted and solubilized from inclusion bodies with guanidine chloride and mechanical treatment.
A first purification step was performed under denaturing conditions using Ni-affinity chromatography. His-tagged protein was eluted with increasing imidazole concentration in good yields (Figure 16).
A crucial step during purification procedure is the refolding of the denatured protein in its native structure. As already mentioned, EPO has two structural disulfide bonds that have to be formed during refolding. This process is a bottle neck as wrong disulfide bonds can be formed leading to misfolded or aggregated protein. Therefore a complex composition of the refolding buffer was required. The optimal composition of the refolding buffer was developed by Marina Rubini in our laboratory using a redox
Figure 15 Expression of WT-EPO in E.coli. 15 % SDS-PAGE of the culture before induction of gene expression (0 hr) and after overnight expression (o/n). The EPO band is marked with an arrow. M: Protein marker.
Figure 16 Purification of WT-EPO via IMAC. 15 % SDS-PAGE of the purification of denatured WT-EPO via Ni-NTA beads. Protein was eluted with increasing concentrations of imidazole. M: Protein marker.
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system consisting of reduced and oxidized glutathione and the stabilizing factor L-arginine to ensure a sufficient formation of the disulfide bonds.
After the first purification via IMAC, refolding was prepared by reducing sample volume using centrifugal concentrators. Subsequently, the protein sample was diluted with the refolding buffer to achieve a total concentration of approximately 0.1 mg/ml (approximately with 1:200 dilution).
After incubation at 4°C overnight, precipitated protein was removed via centrifugation and filtration. Sample volume was reduced via centrifugal concentrators and afterwards dialyzed against CM buffer A to prepare EPO for purification via cation exchanger (Figure 17 A).
The purification step after refolding is important to remove misfolded, but soluble protein from correctly folded protein resulting in approximately 0.8 mg pure protein per 1 l expression.
To verify a proper secondary structure of the refolded protein, a CD spectrum was measured, resulting in a typical spectrum for α-helical protein structure (Figure 17 B).
Additionally, mass analysis was performed identifying the expected species (calculated mass: 19314 Da, found mass: 19314 Da, Figure 18).
Figure 17 A Purification of refolded WT-EPO via CM Sepharose. 15 % SDS-PAGE of elution fractions of WT-EPO. B CD spectrum of WT-EPO. 0.2 mg/ml of WT-EPO in phosphate buffer.
wavelength [nm]
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5.1.2 Optimization of EPO expression
EPO contains two structural disulfide bridges. As these bonds cannot be formed in the reducing bacterial cytoplasm, EPO is insolubly expressed in inclusion bodies.
Aside from some advantages of the insoluble expression, like higher purity, a big disadvantage is the in vitro refolding, more precisely the formation of the correct disulfide bonds. During this process, a lot of protein can be lost due to wrong disulfide bridge formation. To circumvent this problem, some efforts were done to express EPO as soluble protein with correct disulfide bridges. Two special E.coli strains, optimized for the cytoplasmic expression of proteins containing disulfide bonds were used for this purpose: Shuffle T7 and Origami B. After transformation with pET11a-WT-EPO, expression was induced with IPTG and finally analysed via SDS-PAGE. To distinguish between soluble and insoluble EPO expression, cell lysis samples were divided in supernatant and pellet.
Unfortunately, for both strains no soluble expression could be detected (Figure 19).
In conclusion, the genetic mutations in this two strains are not sufficient for the proper folding of EPO in vivo.
Figure 18 Mass spectrum of WT-EPO. Mass was determined via TOF MS ES+. Zoom in on the main peak. [M+H]+ calculated: 19314 Da, [M+H]+ found: 19314 Da.
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5.1.3 Production of fluoro-proline-EPO (FP-EPO) in Escherichia coli
Fluorination is a popular method to modify peptides and proteins with the goal to stabilize the molecule. In this first part, incorporation of 4R- and 4S-fluoro proline in WT-EPO will be shown. Eight prolines have to be exchanged by their fluorinated analog. The incorporation was performed via SPI. Therefore a proline-auxotrophic E.coli strain was mandatory (BL21ΔP). Expression of (fully or partly) fluorinated EPO was verified by SDS-PAGE.Using 1 mM 4S-fluoro proline or 1 mM 4R-fluoro proline, only for 4R-fluoro proline EPO expression was detectable. Concentration of 4S-fluoro proline was doubled, but still fluorinated EPO was nearly undetectable (Figure 20 A). 4R-FP-EPO was expressed in good yields and could be purified with the same protocol as for WT-EPO (Figure 20 B).
It was even possible to study the secondary structure of 4R-FP-EPO via CD spectroscopy, demonstrating a proper folding (Figure 21 B). Measurement of the melting curve showed a very weak cooperativity (data not shown). Cooperativity can give a hint about the cohesion of the protein during denaturing processes. Low cooperativity means that protein unfolding proceeds in steps. In contrast, high cooperativity is detected for proteins loosing secondary structure during one single event in the denaturation process.
Figure 19 Test expression of WT-EPO in Origami B (A) and Shuffle T7 (B). 15 % SDS-PAGE of the culture before induction of WT-EPO expression (0 hr) and overnight/ 5hr expression (o/n). The culture was separated in insoluble parts (P) and the soluble components (SN).
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Mass analysis of 4R-FP-EPO identified two prominent species of EPO with seven or with eight 4R-fluoro prolines, respectively (calculated mass for the incorporation of eight 4R-fluoro prolines: 19458 Da, measured mass for the incorporation of eight 4R-fluoro prolines: 19458 Da, Figure 21 A). Therefore it was possible to exchange almost all eight natural prolines by its fluorinated counterparts.
Unfortunately, stability of the fluorinated protein was very low resulting in fast aggregation and abnormal UV spectra.
Formation of the right disulfide bridges is supposed to be a critical step during refolding of EPO. Wrong formation results in a dramatic loss of protein via aggregation, especially in the case of the fluorinated variant 4R-FP-EPO. Therefore DsbC, a disulfide bridge isomerase, was added (two-fold access) in the refolding buffer to support this process. A lower aggregation tendency during refolding was observed when DsbC was present as assessed by centrifugation. Nevertheless, this positive effect was not consistent during freeze-thaw cycles. A CD spectrum of a 4R-FP-EPO sample demonstrated the dramatic loss of protein (data not shown).
Additionally, removal of DsbC from solution was problematic and DsbC contamination was always present.
It can be concluded that the incorporation of 4R-fluoro proline in EPO at all eight positions is possible. Furthermore, the fluorinated protein is able to fold in a proper secondary structure (Figure 21 B). The incorporation of 4S-fluoro proline was not detectable. This phenomena can be explained by the preferred cis-conformation of the 4S-fluoro proline-peptide bond.[91] It is conceivable that this conformation is not
Figure 20 A Test expression of WT-EPO with 4R-F-Pro and 4S-F-Pro via SPI. 15 % SDS-PAGE of the culture before (0 hr) and after (o/n) protein expression. 2 mM 4S-F-Pro or 1 mM 4R-F-Pro were added.
B Purification of 4R-FP-EPO via CM Sepharose. SDS-PAGE of the elution fractions of 4R-FP-EPO. M.
Protein marker.
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preferred by EPO. The trans-conformation of the 4R-fluoro proline-peptide bond seem to be better tolerated by EPO. Furthermore, it cannot be excluded that 4R-fluoro proline is better accepted by the prolyl-tRNA synthetase.
Regrettably, the aim to establish a more stable EPO variant failed, as 4R-FP-EPO is less stable compared to WT-EPO (lower freeze-thaw stability, fast aggregation).
5.1.4 Design of new EPO variants: K-EPO and 1-Pro-EPO
Narhi et al. presented a modified EPO sequence for a more stable EPO derived from E.coli.[92] They exchanged the asparagine at positon 24, 38 and 83 (naturally occurring N-glycosylation sites) for lysine increasing the isoelectric point. Therefore, they could demonstrate a decreased aggregation tendency by maintaining the secondary structure. Furthermore, the prolines at position 121 and 122 were mutated to asparagine and serine, respectively.[92b] With this EPO mutant in hand, they were able to measure the crystal structure of EPO and its receptor (PDB: 1EER). Therefore we decided to use this variant for our further studies. The amino acid sequence of K-EPO is depicted below:
MAPPRLICDS RVLERYLLEA KEAEK24ITTGC AEHCSLNEK38I TVPDTKVNFY AWKRMEVGQQ AVEVWQGLAL LSEAVLRGQA LLVK83SSQPWE PLQLHVDKAV
Figure 21 A Mass spectrum of 4R-FP-EPO (TOF MS ES+). [M+H]+ calculated for the incorporation of eight 4R-fluoro prolines: 19458 Da, [M+H]+ measured for the incorporation of eight 4R-fluoro prolines:
19458 Da. B CD spectrum of 4R-FP-EPO in phosphate buffer.
wavelength [nm]
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SGLRSLTTLL RALR114AQKEAI S121N122SDAASAAP LRTITADTFR KLFRVYSNFL RGKLKLYTGE ACRTGDRHHH HHH
Again, it has to be mentioned that it was worked with an EPO variant, showing a G114 to R114 shift, like mentioned above.
To take this approach to extremes, an additional EPO variant was created, missing all prolines except proline 42. Proline 2 and 3 were substituted with alanine, proline 87 with valine, proline 90 with alanine, and proline 129 with serine. This changes based on the findings of Elliot et al.[93] Elliot showed that single exchange of the above mentioned positions with the appropriate amino acids has little impact on protein structure, except proline 42. We decided to combine all sites in one molecule to study their influence on EPO expression, structure and stability. The amino acid sequence of 1-Pro-EPO is depicted below:
MAA2A3RLICDS RVLERYLLEA KEAEK24ITTGC AEHCSLNEK38I TVPDTKVNFY AWKRMEVGQQ AVEVWQGLAL LSEAVLRGQA LLVK83SSQV87WE A90LQLHVDKAV SGLRSLTTLL RALR114AQKEAI S121N122SDAASAAS129
LRTITADTFR KLFRVYSNFL RGKLKLYTGE ACRTGDRHHH HHH
The genes for both variants (K-EPO and 1-Pro-EPO) were bought with a codon-optimized sequence and cloned in pET11a with the introduced NdeI and BamHI restriction sites. A test expression was performed in BL21ΔP. As BL21ΔP has no T7 RNA polymerase, pTARA[94] (encoding for a T7 RNA polymerase) was co-transformed with pET11a-K-EPO/ pET11a-1-Pro-EPO. Same conditions as for WT-EPO were used (Figure 22).
Figure 22 Test expression of K-EPO and 1-Pro-EPO in BL21ΔP. 15 % SDS-PAGE of the culture before (0 hr) and after (6 hr) protein expression. EPO band is marked with an arrow.
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A test expression could show that K-EPO could be expressed in good yields. 1-Pro-EPO was expressed in much lower yields. Purification was performed as described for WT-EPO. As it can be seen in Figure 23, K-EPO as well as 1-Pro-EPO can be isolated, solubilized and purified via IMAC. Unfortunately, 1-Pro-EPO aggregated during refolding and the following purification, therefore it was not possible to isolate the protein in adequate amounts. K-EPO was successfully refolded and purified (Figure 24 A) in good yields (approximately 2 mg from 1 l culture). The proper secondary structure of the protein was examined via CD spectroscopy in comparison with WT-EPO (Figure 24 B).
Figure 23 Purification of 1-Pro-EPO and K-EPO via IMAC. 15 % SDS-PAGE of the purification of denatured protein via Ni-NTA beads. Protein was eluted with increasing concentration of imidazole. FT:
flowthrough, W: wash, M: Protein marker.
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5.1.5 Different refolding strategies for K-EPO
As already mentioned several times, refolding of EPO is an important, but inefficient process. It is fundamental for the following studies of the protein. Therefore, several efforts were done to improve the refolding process using L-arginine and a GSH/GSSH redox system.
The first approach to improve the refolding step by addition of DsbC in the refolding buffer was already mentioned above (see Expression of fluoro-proline-EPO (FP-EPO) in Escherichia coli). Although refolding was supported, removal of DsbC was problematic and EPO was not stable during freeze-thaw cycles.
In a second step, a refolding protocol of Boissel et al.[95] was tested using 40 µM copper sulfate and 2% lauroylsarcosine. Lauroylsarcosine is a strong anionic detergent with some similarities to SDS. The denatured protein was dialyzed overnight against the CuSO4 + lauroylsarcosine refolding buffer, followed by a dialysis against Tris buffer. A dramatic protein precipitation was observed in the second dialysis step (data not shown). Therefore, the protein was directly diluted in the CuSO4 + lauroylsarcosine refolding buffer, followed by standard procedures. A highly stable protein could be purified, not precipitating even after stored for weeks at room temperature.
Unfortunately, it was not possible to remove lauroylsarcosine completely, resulting in problems in protein analytics and data interpretation. Especially CD spectroscopy was not possible as the voltage occurred during measurement was way too high. In
Figure 24 A Purification of K-EPO via CM Sepharose. 15 % SDS-PAGE of the elution fractions of K-EPO.
W: wash. B CD spectrum of WT-EPO (black line) and K-EPO (red line) in phosphate buffer. M: Protein marker, W: wash.
wavelength [nm]
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an additional test it could be shown that lauroylsarcosine is the disruptive factor. CD spectra of buffer or protein solution (EPOR), with or without dissolved lauroylsarcosine, were measured showing a clear connection between lauroylsarcosine and high voltage (data not shown). Therefore, it was decided to skip this approach, as it could not be excluded that EPO is not properly folded, but only in solution because of the strong interaction between its hydrophobic parts and lauroylsarcosine. Furthermore, effects of the remaining copper and lauroylsarcosine on biological activity are possible.
Finally, refolding of EPO on Ni-NTA beads was tested. The idea was to avoid a crowded environment leading to aggregation. In a first approach, the beads were rinsed with decreasing concentrations of guanidine chloride and finally EPO was eluted with imidazole. Unfortunately, EPO was aggregating on the beads during washing. In a second approach, Ni-NTA beads with bound EPO were diluted with the GSH/GSSH refolding buffer. Analysis of the beads and the buffer showed that EPO was not bound to the beads, but in solution. The protein was highly impure (data not shown). Therefore, desired beneficial effect of the beads could not be confirmed.
5.1.6 Biophysical studies of WT- and K-EPO
To verify successful refolding of EPO, biophysical analysis of K-EPO was performed. CD spectroscopy was recorded routinely to ensure a proper secondary structure of every purification batch and EPO variant. Consistent with the results of Narhi et al. a typical α-helix-specific spectrum could be observed for K-EPO (see Figure 24 B).[92a] It can be concluded that refolding and purification of K-EPO led to a properly folded protein.
Additionally, melting curves of WT-EPO and K-EPO were measured. Samples with a concentration of 0.2 mg/ml in phosphate buffer were heated with 0.5°C/min monitoring ellipticity at 220 nm. This process is irreversible, as cooling does not lead to the recovery of the protein structure.[92a] Analysis of measured spectra showed an increased melting point for K-EPO (Figure 25 B), compared to WT-EPO (Figure 25 A) reproducing the already published results of Narhi et al.[92a] Furthermore, good cooperativity was observed for K-EPO showing a sharp sigmoidal shape.[92a] It can be assumed that the native state of K-EPO shows a compact, well-structured folding.[96]
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Denaturant-stability of K-EPO was determined incubating the protein with different concentrations of guanidine chloride. The loss of secondary structure with increasing concentrations of guanidine chloride was monitored via CD spectroscopy at 220 nm.
Fitting the resulting curve, a transition midpoint (50 % unfolded protein) at 1.7 M guanidine chloride is obtained (Figure 26).
Narhi et al. observed a transition midpoint for WT-EPO (from E.coli) of 1.2 M in guanidine chloride and for K-EPO of 3.5 M in urea.[29, 92a] For the fully glycosylated EPO they could observe a transition midpoint of 1.75 M in guanidine chloride.[29] This
Figure 25 A Melting curve of WT-EPO. 0.2 mg/ml of WT-EPO were heated from 20°C to 90°C with 0.5°C/min. Change in ellipticity (θ) was followed at 220 nm. B Melting curve of K-EPO. 0.2 mg/ml of K-EPO were heated from 20°C to 90°C with 0.5°C/min. Change in ellipticity (θ) was followed at 220 nm.
(Correct gene sequence of K-EPO).
Figure 26 Guanidine chloride denaturation K-EPO. K-EPO was incubated in different concentrations of guanidine chloride for 1 hr at room temperature. The decrease in ellipticity was followed at 220 nm via CD spectroscopy.
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experiment showed that the transition midpoint of the E.coli-expressed K-EPO is nearly identical with the transition midpoint of fully glycosylated EPO, demonstrating the potency of bacterially expressed EPO for future therapeutic research.
5.1.7 Incorporation of fluorinated amino acids into K-EPO
With the new variant K-EPO in hand, it was attempted to incorporate a variety of different fluorinated amino acids into EPO in a residue-specific manner, e.g.
fluoro proline, tri-fluoro isoleucine (synthesized by Pia Widder, Bachelor thesis)[97], tri-fluoro leucine, para-fluoro phenylalanine, and tri-fluoro valine. The aim was to improve the stability of EPO in matters of proteolytic and thermal stability.
Different auxotrophic E.coli strains were employed for this approach. For the incorporation of fluoro proline, BL21ΔP was the expression system of choice.
Additional auxotrophic strains were purchased from CGSC (Coli Genetic Stock Center), amongst others AB1255 (Ile-auxotrophic), CAG18431 (Ile/Val/Leu-auxotrophic), CV514 (Leu-auxotrophic), and KA197 (Phe-auxotrophic).
K-EPO has 23 leucine, 11 valine, 6 proline, 4 phenylalanine, and 5 isoleucine residues. The bulk of these amino acids are located in the hydrophobic core of EPO (Figure 27).
Tirrell et al. could show that enhanced IleRS and ValRS activity can improve the incorporation of fluorinated isoleucine or valine analogs into proteins.[98] Therefore we obtained the required genes with their endogenous promotors by PCR from the E.coli genome (sequence information was determined from EcoGene). The introduced restriction sites (for ileS: EcoRI and HindIII, for valS: EcoRI and ClaI) were used to clone the genes in pET11a/K-EPO or pET11a/1-Pro-EPO. Correct cloning was
Figure 27 Cristal structure of K-EPO (PDB: 1EER). Residues attempted to be exchanged by their fluorinated counterparts are marked with different colours.
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verified by sequencing (pET11a/K-EPO/valS; pET11a/K-EPO/ileS; pET11a/1-Pro-EPO/valS; pET11a/1-Pro-EPO/ileS).
With the fluorinated amino acids and the auxotrophic strains in hand, first expression experiments were performed. All auxotrophic strains mentioned above were co-transformed with pTARA and pET11a/K-EPO or pET11a/1-Pro-EPO.
Additionally, AB1255 and CAG18431 were co-transformed with pTARA and pET11a/K-EPO (1-Pro-EPO)/ileS or pET11a/K-EPO (1-Pro-EPO)/valS.
At first, expression of K-EPO and 1-Pro-EPO was tested with natural amino acids in all auxotrophic strains, confirming the successful expression of K-EPO in all systems. 1-Pro-EPO was expressed in none of the strains (data not shown).
Therefore it was decided not to continue with the variant 1-Pro-EPO as the protein is highly instable or not expressed at all. For K-EPO, incorporation with all mentioned fluorinated amino acids was attempted (Figure 28).
Like K-EPO, all fluorinated EPO variants were expressed in inclusion bodies. Using BL21ΔP, incorporation of 4S-fluoro proline in K-EPO could not be detected. 4R-fluoro proline could be introduced in K-EPO in small yields (Figure 28 B). K-EPO with para-fluoro phenylalanine was not expressed, as could be shown in Figure 28 C. The same is true for the incorporation of tri-fluoro valine in the presence or absence of overexpressed ValRS (Figure 28 D). Tri-fluoro isoleucine was incorporated in K-EPO using strain AB1255 overexpressing IleRS (Figure 28 E). Further experiments were performed by Pia Widder during her bachelor thesis.[97] Fluorinated amino acids were used with different concentrations, depending on solubility and toxicity. The experiments shown here are examples with the highest concentrations used.
Incorporation of tri-fluoro leucine, using strain CV514, resulted in satisfying expression of F-Leu-EPO (overnight incubation, Figure 28 A). Same was true for the strain CAG18431 (data not shown).
For further studies, F-Leu-EPO was isolated from inclusion bodies and refolded.
Unfortunately, due to precipitation, fluorinated protein could not be isolated after refolding (data not shown). The same is true for F-Ile-EPO (Pia Widder). Therefore, it was not possible to isolate any of the fluorinated EPO variants to perform stability or activity assays.
As already mentioned in the introduction, fluorination is widely used to stabilize proteins and peptides. In most cases, hydrophobic amino acids are exchanged by their fluorinated counterparts, primary placed in α-helical scaffolds.[99] The stabilizing effect of introduced fluorinated amino acids was mainly explained by increasing
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hydrophobicity in the coiled coil structures.[57c, 57d] Several studies demonstrated that highly fluorinated amino acids have a lower α-helix propensity.[56, 100] Amongst others,
hydrophobicity in the coiled coil structures.[57c, 57d] Several studies demonstrated that highly fluorinated amino acids have a lower α-helix propensity.[56, 100] Amongst others,