Engineering of bacterially expressed
Erythropoietin by means of non-natural amino acids for improved therapeutic potential
Dissertation submitted for the degree of Doctor of Natural Sciences
(Dr. rer. nat.)
Presented by Eugenia Hoffmann
at the
Faculty of Sciences Department of Chemistry
Date of the oral examination: 07.11.2016 First referee: Dr. Marina Rubini Second referee: Prof. Dr. Andreas Marx
Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-0-375490
University of Konstanz. It was funded by a scholarship of the “Graduate School of Chemical Biology”, University of Konstanz for 3 years.
E. Hoffmann, K. Streichert, N. Nischan, C. Seitz, T. Brunner, S. Schwagerus, C. P.
Hackenberger, M. Rubini, Molecular bioSystems 2016, 12, 1750-5.
Danksagung
Zunächst möchte ich mich bei Dr. Marina Rubini bedanken, dass sie mir die Möglichkeit gegeben hat meine Doktorarbeit in ihrer Gruppe durchzuführen, sowie für die Bereitstellung des sehr interessanten Themas. Ich habe nicht nur thematisch und methodisch sehr viel gelernt, sondern habe auch eine sehr gute Freundin und Mentorin gefunden.
Des Weiteren bedanke ich mich bei Prof. Dr. Andreas Marx, dass ich nicht nur aktiv am Leben seiner Arbeitsgruppe teilnehmen durfte, sondern auch vollen Zugang zur Laboreinrichtung hatte. Außerdem danke ich Herrn Marx für die Übernahme des Zweitgutachtens und die Mitgliedschaft in meinem Thesis-Komitee.
Ferner bedanke ich mich bei Prof. Dr. Wolfram Welte für die Mitgliedschaft in meinem Thesis-Komitee.
Prof. Dr. Martin Scheffner danke ich für die Übernahme des Prüfungsvorsitzes.
Ich bedanke mich außerdem bei der gesamten AG Marx für die tolle Arbeitsatmosphäre und Hilfe bei allen Fragestellungen. Insbesondere bedanke ich mich bei Katharina Streichert für die gute Zusammenarbeit und bei meiner Masterstudentin Lilian Karl für eine tolle Zeit. Bei Stephan Hacker, Matthias Drum, Eva Höllmüller und Vlasta Radusevic bedanke ich mich für ihre Freundschaft und fröhliche Art.
Bei Eva Höllmüller, Daniel Rösner, Moritz Schmidt und Janina von Watzdorf bedanke ich mich für die ausführlichen und hilfreichen Kommentare zur Verbesserung der schriftlichen und graphischen Ausführungen dieser Arbeit.
Insbesondere möchte ich Janina von Watzdorf danken, die nicht nur das gesamte Studium sondern auch während der Promotion zu einer festen Stütze geworden ist, deren Freundschaft ich nicht mehr missen möchte. Ich hoffe unsere Wege werden sich noch häufig kreuzen.
Für ihre Freundschaft und die vielen tollen Momente während und nach dem Studium möchte ich mich bei Linda Holst, Moritz Schmidt, Timo Griesinger und Matthias Schneiderhan bedanken.
Dr. Marilena Manea danke ich für ihre Hilfe bei fachlichen Fragestellungen und experimentellen Durchführungen.
Meinen Eltern, meiner Schwester, meinem Bruder und der gesamten Familie möchte ich für ihre unerschütterliche Unterstützung in schweren und schönen Tagen während dem Studium und der Promotion danken.
Meinem Freund Andreas danke ich für die unglaublich schönen Momente und die Hilfe in jeder Lebenslage, für seine Inspiration, seinen Mut und seinen Optimismus.
Ich freue mich auf die nächsten 100 Jahre….
Content
1. SUMMARY ... 2
2. ZUSAMMENFASSUNG ... 6
3. INTRODUCTION ...10
3.1 Erythropoietin ... 10
3.2 Glycosylation of proteins ... 12
3.3 Recombinant EPO in medicine ... 13
3.4 PEGylation of biopharmaceuticals ... 14
3.5 Protein synthesis in cells ... 15
3.6 Fluorinated amino acids ... 18
3.7 Engineering and expanding the genetic code ... 19
3.8 Chemical modification of proteins ... 22
4. AIM OF THE THESIS ...28
5. RESULTS AND DISCUSSION ...30
5.1 Production of EPO and incorporation of fluorinated amino acids ... 30
5.1.1 Expression and purification of WT-EPO in Escherichia coli ... 30
5.1.2 Optimization of EPO expression ... 33
5.1.3 Production of fluoro-proline-EPO (FP-EPO) in Escherichia coli ... 34
5.1.4 Design of new EPO variants: K-EPO and 1-Pro-EPO ... 36
5.1.5 Different refolding strategies for K-EPO... 39
5.1.6 Biophysical studies of WT- and K-EPO ... 40
5.1.7 Incorporation of fluorinated amino acids into K-EPO ... 42
5.1.8 DSB-K-EPO: An EPO variant with only one Disulfide Bridge ... 44
5.2 Incorporation of para-azido phenylalanine at defined positions in EPO ... 47
5.2.1 Incorporation of pAzF in EPO: Gene design ... 48
5.2.2 Expression of K-EPOpAzF in E.coli and protein purification ... 49
5.2.3 Staudinger Phosphite Reaction (SPR) of K-EPOpAzF ... 49
5.2.4 CD spectra of PEGylated and non-PEGylated K-EPO variants ... 56
5.2.5 Melting curves of PEGylated and non-PEGylated EPO variants ... 58
5.2.6 Solubility-tests of PEGylated and non-PEGylated K-EPOpAzF ... 60
5.2.7 Proteolytic stability of PEGylated and non-PEGylated K-EPOpAzF ... 62
5.2.8 Doubly PEGylated K-EPO ... 63
5.2.9 Threefold PEGylated K-EPO ... 64
5.2.10 In vitro activity of modified K-EPO ... 66
6. OUTLOOK ... 72
7. MATERIALS AND METHODS ... 74
7.1 General Methods in Microbiology and Biochemistry ... 74
7.2 Expression of EPO in Escherichia coli, protein purification and analysis; Incorporation of fluorinated amino acids in EPO ... 79
7.3 Synthesis of PEG phosphite; Incorporation of para-azido phenylalanine in EPO and the Staudinger Phosphite Reaction ... 84
7.4 In vitro assays: cell culture experiments ... 87
7.5 Chemicals, amino acids and antibiotics ... 92
7.6 Plasmids ... 94
7.7 Bacterial and human cell lines ... 95
7.8 Antibiotics for bacterial selection ... 96
7.9 Restriction enzymes, polymerases, ligases, proteins and nucleotides ... 97
7.10 FPLC columns, batch purification materials, Kits ... 97
7.11 Marker... 98
7.12 Cuvettes... 98
7.13 Consumables in microbiology ... 98
7.14 Consumables in the cell culture: media, buffer, solutions ... 99
7.15 Consumables in the cell culture: plastics, plates, etc. ... 99
7.16 Equipment in microbiology ... 100
7.17 Equipment in cell culture ... 101
7.18 Buffers and Media ... 102
7.18.2 Growth media ... 104
7.18.3 Buffer for bacterial lysis, extraction and Ni-NTA purification... 105
7.18.4 Refolding buffer ... 106
7.18.5 Other buffers for different applications ... 106
7.18.6 Buffers for cell culture ... 107
8. SEQUENCES ... 110
9. REFERENCES ... 120
10. LIST OF ABBREVIATIONS ... 126
2
1. Summary
The main incentive for the development of new techniques and biological tools is to understand nature or even improve it. In this thesis, approaches were evolved to realize this aim, working with the therapeutic protein Erythropoietin (EPO). EPO is highly glycosylated, hence stability, solubility and functionality of the protein depends on this posttranslational modification. Due to this glycosylation, production of EPO for therapeutic approaches has to be performed in eukaryotic systems, making EPO very expensive. As glycosylation is template-independent, glycosylation patterns can be very heterogeneous depending on the expression system and conditions.
A complementary approach to stabilize EPO was investigated in the first part of this thesis. Incorporation of non-natural fluorinated amino acids into the protein backbone of EPO should clarify whether EPO is still properly folded and more resistant towards proteases and thermal denaturation like it was shown for other proteins and peptides. EPO was expressed in Escherichia coli (E.coli). As E.coli is not able to glycosylate proteins, isolated EPO is completely deglycosylated and must be recovered and refolded from inclusion bodies. Incorporation of fluorinated amino acids was performed via Selective Pressure Incorporation using auxotrophic bacterial strains. A variety of different hydrophobic, fluorinated amino acids were tested for incorporation into the backbone of EPO as well as evaluated for their influence on protein folding and stability. Unfortunately, the majority of the tested fluorinated amino acids could not be incorporated into EPO, or the synthesized protein was immediately degraded in cell. Nevertheless, EPO modified with fluorinated isoleucine could be expressed and purified. Refolding into its native structure and following purification led to complete loss of the fluorinated protein. It can be assumed that the incorporation of the fluorinated amino acids not only disturbed secondary and tertiary structure formation but also increased the hydrophobicity of the protein. As EPO already has a very hydrophobic surface, usually covered by glycans, increase in surface hydrophobicity is probably the reason for aggregation. However, enhanced resistance towards proteases and temperature, arising from increased core hydrophobicity, could not be studied.
In the second part of this work EPO was again modified with a non-natural amino acid, para-azido phenylalanine (pAzF). In contrast to the first part, stabilization of EPO
Summary
3
should not be based on the non-natural amino acid, but on the following site-specific modification with a simple glycan mimic, polyethylene glycol (PEG). The site-specific PEGylation of un-glycosylated EPO does not only give us the opportunity to study the impact of the different modification sites on protein stability, but should also establish an additional potent method for post-translational modification and stabilization of proteins for therapeutic approaches.
EPO has four naturally occurring glycosylation sites at position 24, 38, 83 ( N-glycosylation) and 126 ( O-glycosylation). These positions were chosen to introduce pAzF via Amber Stop Codon Suppression using an evolved Methanococcus jannaschii tyrosyl -tRNA synthetase/tRNA pair. As expression system E.coli was chosen. Subsequent PEGylation of these sites with branched PEG resulted in different PEGylated EPO variants. Incorporation of pAzF at a single site in EPO could be shown with high efficiency. Refolding of pAzF-modified EPO in its native structure already gave first hints on the influence of pAzF on protein stability. EPO modified at position 24, 38 and 126 showed a nearly unaltered behaviour in folding, whereas incorporation of pAzF at position 83 led to complete destabilization of EPO after refolding. Nevertheless, site-specific PEGylation of EPOpAzF via Staudinger Phosphite Reaction before protein refolding could not only rescue the protein from aggregation, but even enhanced protein stability against unspecific aggregation at physiological temperature and proteases. In vitro assays demonstrated the preserved receptor binding and activation potency of all PEGylated variants. This is true for the shorter (30 PEG units in total) as well as for the longer (90 PEG units in total) PEG chains used in this study. In vitro assays showed that modification at position 24 or 38 has similar improving effects on activity compared to fully glycosylated recombinant EPO expressed in Chinese hamster ovary (CHO) cells. EPO PEGylated at position 83 or 126 showed a high activity.
Incorporation of three non-natural amino acids (pAzF) in one EPO molecule could be demonstrated allowing the PEGylation of all naturally occurring N-glycosylation sites. The EC50 (half effective concentration) value was lower than for the single PEGylated variants, but maximal cell proliferation was nearly as good as for the fully glycosylated positive control. It can be concluded that extensive PEGylation may influence receptor binding, but protein solubility and resistance is highly increased resulting in a prolonged phase of active protein in solution.
In summary, we could successfully modify all naturally occurring glycosylation sites of EPO (single, double and triple PEGylation) with two different PEG moieties. All generated variants showed increased stability and in vitro activity compared to non-
4
modified EPO, demonstrating the high potential of this approach. Analysing the different modification sites, position 24 and 38 seem to have similar impact on stability and activity compared to the fully glycosylated variant. But position 83 showed high impact of pAzF incorporation, as well as of modification with the different PEG chains.
It could be demonstrated that the longer the PEG chains, the better the in vitro activity.
On the other hand, incorporation of pAzF at position 126 is less problematic, although we could again detect an impact of PEG length on biological activity with better EC50
values for the longer PEG chains. These results are in good agreement with literature, as they show that N-glycosylation sites have great impact on EPO activity.
Restrictively, it has to be mentioned that the influence of pAzF incorporation into EPO is not neutral.
The three-fold PEGylated variant is a big success, as not only the incorporation of three non-natural amino acids in one molecule is very challenging but also the isolation of the fully PEGylated molecule is not trivial. It has the potential to replace the common, random modification of therapeutic proteins at all e.g. lysine residues to allow full control over modification and biological function.
6
2. Zusammenfassung
Das Verständnis über die Natur und die Verbesserung dieser ist die treibende Kraft der Wissenschaft neue Technologien und Materialien zu entwickeln. In dieser Arbeit werden, basierend auf dem therapeutischen Protein Erythropoietin (EPO), Ansätze entwickelt um diese Ziele zu erreichen. EPO trägt vier Glykosylierungsketten, die sowohl für die Stabilität als auch für die Aktivität des Proteins im menschlichen Körper unerlässlich sind. Aufgrund dieser Glykosylierungen wird therapeutisches EPO in eukaryotischen Zelllinien produziert. Das macht das Medikament sehr teuer.
Im ersten Teil dieser Arbeit wurden nicht-natürliche, fluorierte Aminosäuren in EPO eingebaut. Es sollte geklärt werden, ob das Protein sich weiterhin in seine native Form rückfaltet und erhöhte Toleranz gegenüber Temperatur und Proteasen zeigt, wie bereits für andere Proteine und Peptide gezeigt wurde. In dieser Arbeit wurde EPO ausschließlich in E.coli exprimiert. Da E.coli nicht in der Lage ist Proteine zu glykosylieren, handelt es sich bei dem isolierten Protein um eine vollständig deglykosylierte Form. Die Einführung der fluorierten Aminosäuren in EPO wurde mittels „Selective Pressure Incorporation“ realisiert. Verschiedene hydrophobe, fluorierte Aminosäuren wurden auf ihre Fähigkeit getestet in das Proteinrückgrat eingebaut zu werden. Außerdem wurde ihr Einfluss auf die Proteinstabilität und Proteinfaltung untersucht. Leider konnte der Großteil der untersuchten Aminosäuren nicht in das Protein eingebaut werden, oder das synthetisierte Protein wurde in der Zelle direkt abgebaut. EPO, modifiziert mit fluoriertem Isoleucin konnte jedoch in ausreichenden Mengen exprimiert und unter denaturierenden Bedingungen isoliert werden. Die Rückfaltung in die native Struktur und anschließende Reinigung führten jedoch zum kompletten Verlust des Proteins. Es kann daraus geschlossen werden, dass der Einbau der fluorierten Aminosäuren nicht nur die Ausbildung der Sekundär- und Tertiärstruktur behindert, sondern auch zur Erhöhung der Hydrophobizität führt.
Da EPO von Natur aus eine hydrophobe Oberfläche aufweist, die normalerweise von den Glykanketten verdeckt wird, könnte eine weitere Zunahme der Hydrophobizität die verstärkte Aggregation erklären. Aufgrund dieser Problematik konnten keine Untersuchungen bezüglich der Stabilität gegen Proteasen und erhöhter Temperatur, hervorgerufen durch eine mögliche Erhöhung der Kernhydrophobizität, durchgeführt werden.
Summary
7
Im zweiten Teil dieser Arbeit wurde EPO mit der nicht-natürlichen Aminosäure para-Azidophenylalanine (pAzF) modifiziert. Im Gegensatz zum ersten Teil der Arbeit sollte die Stabilisierung von EPO nicht durch den Einbau der nicht-natürlichen Aminosäure erfolgen, sondern durch die anschließende, ortsspezifische Modifikation mit einem simplen Glykanketten-Ersatz, dem Polyethylenglykol (PEG). Wie bereits oben erwähnt, ist EPO vierfach glykosyliert. Diese Glykosylierung ist von herausragender Bedeutung für die biologische Aktivität von EPO in lebenden Systemen. Andererseits stellt diese Glykosylierung ein Problem in der Medikamentenproduktion dar, da sie nur in anspruchsvollen Säugetierzellen funktionell nachgebildet werden kann. Erschwerend kommt hinzu, dass das Glykosylierungsmuster sehr heterogen sein kann und stark von kleinsten Änderungen in den Expressionsbedingungen abhängt. Die positionsabhängige und spezifische PEGylierung von unglykosyliertem EPO gibt uns nicht nur die Möglichkeit den Einfluss der einzelnen Modifikationsstellen auf die Stabilität des Proteins zu untersuchen, sondern soll auch eine weitere wirksame Methode zur posttranslationalen Modifikation und Stabilisierung von therapeutischen Proteinen aufzeigen.
EPO hat vier natürlich vorkommende Glykosylierungsstellen. An Position 24, 38 und 83 findet sich eine N-Glykosylierung und an Position 126 eine O-Glykosylierung.
Diese Positionen wurden ausgewählt und pAzF mittels „Amber Stop Codon Suppression“ eingebaut. Die anschließende PEGylierung an diesen Stellen mit verzweigten PEG-Ketten führte zu einer Reihe von verschiedenen PEGylierten EPO- Varianten. Es konnte gezeigt werden, dass der Einbau von pAzF an einer einzigen Stelle in EPO zu guten Ausbeuten an unlöslichem Protein führt. Die Rückfaltung von pAzF-haltigem EPO in seine native Struktur gab erste Hinweise auf den Einfluss der nicht-natürlichen Aminosäure auf die Stabilität des Proteins. Der Einbau von pAzF an Position 24, 38 und 126 hatte keinen signifikanten Einfluss auf die Rückfaltung und Stabilität des Proteins. Wurde pAzF an Position 83 eingebaut, kam es zur vollständigen Aggregation des Proteins während der Rückfaltung. Interessanterweise konnte eine PEGylierung mittels Staudinger Phosphit Reaktion vor der Rückfaltung des Proteins diese Aggregation verhindern und das Protein sogar resistenter gegenüber Proteasen und erhöhter Temperatur machen. In vitro Tests an humanen Zelllinien konnten zeigen, dass die Rezeptorbindung und -aktivierung von EPO durch die PEGylierungen an allen untersuchten Stellen nicht nennenswert eingeschränkt wurde. Dies wurde sowohl für die kürzere PEG-Variante (mit insgesamt 30 PEG- Einheiten) sowie für die längere PEG-Variante (mit insgesamt 90 PEG-Einheiten) gezeigt. PEGylierung an Position 24 oder 38 haben in den in vitro Tests zu
8
vergleichbaren Resultaten geführt. Jedoch zeigten die Modifikationen an Position 83 oder 126 erhöhte Aktivität.
Abschließend gelang es drei nicht-natürliche Aminosäuren (pAzF) in ein einziges EPO-Molekül einzubauen. Dies ermöglichte die PEGylierung aller natürlich vorkommenden N-Glykosylierungsstellen (Position 24, 38 und 83). Obwohl der EC50- Wert (effektive Konzentration) höher war als für die einfach-PEGylierten Varianten, konnte eine sehr gute maximale Zellproliferation ermittelt werden, vergleichbar mit der Positivkontrolle, also vollglykosyliertem EPO. Daraus kann geschlossen werden, dass die dreifache PEGylierung von EPO durchaus die Rezeptorbindung beeinflussen kann, jedoch ist sowohl die Löslichkeit als auch Stabilität des Proteins soweit verbessert, dass es länger als lösliches, aktives Protein vorliegt.
Zusammenfassend kann gesagt werden, dass es gelungen ist alle natürlich vorkommenden Glykosylierungsstellen von EPO mit zwei verschiedenen PEG- Varianten zu modifizieren (einfach-, zweifach-, dreifach PEGylierung). Alle generierten Varianten zeigen erhöhte Stabilität und in vitro Aktivität im Vergleich zu unPEGylierten Proteinen, was das Potenzial dieser Modifizierungsmethode aufzeigt.
Betrachtet man die einzelnen Modifizierungsstellen, so hat die PEGylierung an Position 24 oder 38 einen ähnlich positiven Einfluss auf Proteinstabilität und Aktivität im Vergleich zum vollglykosylierten Protein. Andererseits ist der Einbau von pAzF an Position 83 und die PEGylierung mit den verschiedenen PEG-Varianten mit immensen Änderungen in der Proteinstabilität und Aktivität verbunden. Der Einbau von pAzF an Position 126 ist unproblematisch, wobei auch hier, so wie für Position 83, eine erhöhte Aktivität mit längerer PEG-Kette ermittelt wurde. Die dreifach- PEGylierte EPO-Variante ist ein großer Erfolg, da der Einbau von drei nicht- natürlichen Aminosäuren in EPO und die Isolierung der dreifach-modifizierten Proteine nicht trivial und mit großen Hindernissen verbunden ist. Dieser Ansatz hat das Potenzial die bisher gebräuchliche, zufällige Modifikation von therapeutischen Proteinen durch eine kontrollierbare Methode abzulösen.
9
10
3. Introduction
3.1 Erythropoietin
The presented thesis focuses on one of the most important drugs: Erythropoietin (EPO). The physiological function of EPO is the stimulation of red blood cell proliferation and maintenance of a constant haemoglobin level in blood.[8]
EPO is a 30.4 kDa glycoprotein. In its mature form it has 165 amino acid residues and 40 % of the mass contribute from the carbohydrate moieties.[9] The human EPO mRNA encodes for a polypeptide of 193 amino acids. After cleavage of the leader sequence and glycosylation in the Golgi apparatus, EPO is in its active form.[10] EPO is glycosylated at four positions: Asn24, Asn38 and Asn83 ( N-glycosylation), and at Ser126 ( O-glycosylation). This extensive glycosylation has several functions, amongst others the protection against proteases, increase of the hydrodynamic radius and the manipulation of the EPO receptor binding properties.[11]
Figure 1 Cristal structure of EPO and its receptor. EPO (in yellow) leads to the dimerization of its receptor (in blue) (PDB file: 1EER).
Introduction
11
The protein scaffold consists of four anti-parallel α-helices, two β-sheets and two intramolecular disulfide bonds (Cys7-Cys161 and Cys29-Cys33) as solved by NMR and X-ray (Figure 1).[9a, 12] In adults, EPO is expressed in the kidney, regulated by the hypoxia-inducible transcription factors (HIFs). Therefore, chronic kidney disease lead to EPO deficiency.[13]
The EPO receptor (EPOR) is mainly located in the bone marrow on erythroid progenitors as preformed homodimer.[14] The receptor is a membrane-spanning 59 kDa glycoprotein with 484 amino acid residues.[15] Binding of EPO to the receptor dimer leads to conformational changes of EPOR and intracellular activation of the JAK-2 pathway resulting in the maturation of new red blood cells.[16] EPO has two receptor binding sites with different affinities to the receptor: Kd1 ≈ 1 nM and Kd2 ≈ 1 µM.[17] After activation, the EPO/EPOR complex is internalized. 40 % of EPO are degraded in the proteasome, 60 % are re-secreted. This process is one part to maintain EPO equilibrium in blood.[16]
Figure 2 summarizes the activation pathway of EPO. With decreasing oxygen concentration in the blood, EPO expression in kidney is induced. The produced EPO protein leads to the maturation of erythrocytes by binding to its receptor in the progenitor cells in the bone marrow. With the increasing erythrocyte number, oxygen level in the blood is rising.[6]
Figure 2 Activation pathway of EPO. With decreasing oxygen levels, EPO expression in the kidney is induced. Increased EPO concentration leads to the maturation of erythrocytes in the bone marrow. The oxygen level is rising. Adapted by Bunn et al.[6]
12
3.2 Glycosylation of proteins
Glycosylation of proteins is one of the most prominent post-translational modifications. It is estimated that nearly half of the human proteins are glycosylated.[18]
The glycosylation pattern provides an additional level of information storage on the molecular level, as changes in glycosylation can modify the function of a protein.[19]
The influences of protein glycosylation are versatile, starting with its influence in protein secretion, protein folding and its participation in quality control in the endoplasmic reticulum.[20] Furthermore, glycosylation is crucial for protection of the protein against proteases, increase of solubility, prevention against fast clearance and immune surveillance.[21]
In mammals, nearly 700 proteins contribute to the production of the highly complex and diverse glycans, consisting of only few monosaccharides: fucose (Fuc), galactose (Gal), glucose (Glc), N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc, GN), glucuronic acid (GlcA), iduronic acid (IdoA), mannose (Man), sialic acid (SA) and xylose (Xyl).[19, 22] Glycans can be attached to proteins in different ways.
Coupling to an Asn side chain leads to the formation of an amide linkage (N- glycosylation). The consensus sequence for this process is Asn-Xxx-Ser/Thr (Xxx:
any amino acid, except Pro). Covalent binding to a Ser or Thr side chain leads to the formation of a glycosidic linkage (O-glycosylation).[21b, 23]
EPO has three N-glycosylation sites and one O-glycosylation site. The tetra- antennary species of the N-glycans is most prominent in recombinant EPO expressed in Chinese hamster ovary (CHO) cells, but also other species can be found.[24] The bi-antennary and tri-antennary structures are found to be responsible for a variation in biological activity (N-glycosylation).[24] Figure 3 shows the schematic structure of common N-glycans of EPO.[24b] This microheterogeneity is not only due to different branches, but also because of different sialic acid contents.[25]
Studies demonstrated that the carbohydrate content influences the affinity of EPO to its receptor. Furthermore it was shown that glycosylated EPO has an increased circulation period.[26] As the peptide backbone of EPO is very hydrophobic, glycosylation is an important factor to keep the protein in solution, reduce aggregation events and shield potential immunogenic surfaces.[25a]
Introduction
13
Already very early it was found that sialic acid residues play a crucial role for the in vivo activity of EPO. Desialylation of EPO leads to the complete loss of its activity
[11b, 24a] because asialo-EPO is trapped in the liver.[27] Dubé et al.[28] studied the loss of glycosylation at different glycosylation sites. They could show that deglycosylation at position 38, 83 and 126 cause a rapid degradation of EPO (expressed in baby hamster kidney (BHK) cells). EPO deglycosylated at position 24 was secreted without any restrictions.[28] Narhi et al.[29] demonstrated that the presence of the glycans has a dramatic impact on the stability of EPO against denaturing conditions and thermal denaturation. Sialic acid has no influence on these properties.[29] The biological effect of the O-glycosylation Ser 126 is less extensively studied. Ochi et al. demonstrated that the absence of the O-glycosylation has no influence on in vivo activity.[30]
3.3 Recombinant EPO in medicine
EPO is one of the best-selling drugs world-wide, used for the treatment of anaemia caused by chronical kidney disease, HIV, chancer, etc.[6] Currently, different variants are on the market. A uniform nomenclature should guide through the different molecules. Epoetin is an international name for eukaryotic cell-derived recombinant human EPO (rhEPO) with an unmodified amino acid sequence. Modifications in the amino acid sequence are indicated with a random prefix.[31] Greek letters stand for different glycosylation patterns. The most prominent provider of rhEPO are Chinese hamster ovary cells (CHO). Some famous representatives are Epogen® from Amgen (Epoetin α) or NeoRecormon® from F.Hoffmann-La Roche (Epoetin β). [13a, 31]
After patents expired, further drugs were placed on the market (biosimilars), e.g.
Binocrit® from Sandoz (Epoetin α) or Retacrit® from Hospira (Epoetin ζ).[31]
Figure 3 N-glycans commonly found in recombinant EPO derived from CHO cells as well as in human EPO. Fucosylation is highly prominent in both variants.[1]
14
Furthermore, the protein was modified to prolong its half-life in blood. One of this so called “biobetters” is Arnesp® from Amgen. It has two additional glycosylations and thereby five exchanges in amino acid sequence (Darbepoetin α, ≈ 37 kDa).[11a]
Mircera® from F.Hoffmann-La Roche has, besides the natural glycosylation, an additionally attached PEG moiety (PEG: polyethylene glycol, PEGylation) increasing the molecular mass to 60 kDa (methoxy PEG-epoetin β).[31]
3.4 PEGylation of biopharmaceuticals
The PEGylation of proteins and peptides is a widely used method to modify properties of macromolecules. PEG is a simple, chemically inert polymer composed of a variable number of polyethylene glycol units. Therefore, molecular mass of PEG can be very diverse, starting with a few Da to several hundred kDa (Figure 4). PEG is supposed to be non-biodegradable, even though oxidized species can be found in biological matrices.[32]
Although polypeptides are very promising and potent drugs, they bear several drawbacks. First of all, they can be digested by proteases, aggregate, induce immune responses or they can be cleared from blood in the kidney. Renal clearance is mainly involved in the elimination of hydrophilic proteins, depending on size and charge of the molecule.[33] In summary, they often have a short half-life in blood.[34]
PEGylation was found to be a solution for these problems, improving pharmacokinetics and pharmacodynamics of polypeptide drugs. As PEG is highly water soluble, PEGylation of proteins improve their solubility. Additionally, large PEG chains increase the molecular mass and the hydrodynamic radius of the PEG-protein conjugate and therefore decrease renal clearance. Shielding of the protein against proteases is another important property of a PEG modification.[34] Furthermore, PEGylated proteins seem to be more stable against temperature and pH oscillations.[35] Lee and colleagues[36] found that the modification of a protein with few long PEG chains is more effective in protection than a modification with many short chains.
Figure 4 Methoxy polyethylene glycol (mPEG) with 45 ethylene glycol units (≈ 2 kDa).
Introduction
15
Although some in vitro assays showed a decrease in activity of PEGylated proteins (e.g. due to modification of receptor binding), in vivo activity is predominantly increased, because of longer protein half-life in blood.[37]
PEG is considered to be non-immunogenic, although several publications claim the immunogenic potential of PEGylated drugs, identifying anti-PEG antibodies in treated organisms.[38] On the other hand, studies showed that PEGylation suppresses immunogenicity and antigenicity.[39] Especially branched PEG chains seem to have a positive effect on proteolytic stability and antigenicity.[40] In summary, the science community does not agree on the effect of PEG on the immune response.
The coupling of PEG moieties to a polypeptide can be performed with different strategies. A widely used method is the random conjugation to ε- and α- amino groups of a protein.[34, 41] This and further modification strategies will be discussed elsewhere in this thesis (3.8 Chemical modification of proteins).
3.5 Protein synthesis in cells
The process of protein synthesis ( translation) is of great interest in this thesis, as it is the basis for the incorporation of non-natural amino acids in a protein sequence. Therefore, a short introduction about the general procedure of this complex mechanism should facilitate the understanding of the following chapters.
Translation, the generation of a polypeptide chain on the basis of an mRNA molecule, is a pillar of the central dogma of molecular biology. This dogma states that the genetic information, stored in the DNA is transcribed in RNA and then translated in a protein (Figure 5).[42]
Translation takes place at the ribosome. In eukaryotes, this multi-protein complex is located in the cytosol and on the surface of the endoplasmic reticulum (ER). As prokaryotes do not have a nucleus, transcription and translation are performed
Figure 5 Scheme of the central dogma of molecular biology.
16
parallel in the cytosol. The general mechanism of translation is similar in eukaryotic and prokaryotic cells, therefore it will be focused on the prokaryotic system.
Aminoacylation of transfer RNA
Before starting with the protein synthesis at the ribosome, one important upstream step will be discussed in more detail: the aminoacylation of transfer RNA (tRNA) with its cognate amino acid.
The tRNA is the connection between the coding triplet on the mRNA and the corresponding amino acid. Already in 1955, Zamecnik et al. identified a covalent linkage between amino acids and RNA.[43] 20 natural amino acids have to be transported to the ribosome in response to a defined coding triplet. As the genetic code is redundant, there are several triplets coding for the same amino acid, but there are much less tRNA molecules. To overcome this problem, the anticodon of the tRNA, interacting with the triplet of the mRNA, has a special “wobble-position” allowing the interaction with different codons (Figure 6).[44]
The aminoacylation is the covalent attachment of an amino acid to its tRNA, catalysed by the aminoacyl-tRNA-synthetase (aaRS). Every amino acid has one or even more specific aaRS and tRNAs to ensure a precise decoding of the mRNA.[45]
There are two different classes of aaRS, differing in size, structure and the active site.[46] Furthermore, they show a different linkage profile: Class I enzymes couple the amino acid at the 2`-hydroxy group of the terminal tRNA-adenosine, whereas class II
Figure 6 Codon-anticodon pairing of a leucyl-tRNA. An aminoacylated tRNA binds to the corresponding codon on mRNA with a normal base pairing (left site). The same leucyl-tRNA shows a wooble pairing for the second leucine anticodon. Inspired by P. Russel.[4]
Introduction
17
enzymes couple to the 3`-hydroxy group.[47] In general, aminoacylation can be divided in two reactions. First of all, the aaRS binds its cognate amino acid and activates it in the presents of ATP, resulting in an aminoacyl-adenylate and pyrophosphate. The second step is the nucleophilic attack of the ribose 2`- or 3`- hydroxyl group of the 3`- adenosine-tRNA.[48]
Concluding the upper section, aminoacylation is a significant factor to ensure the fidelity of the genetic code. Therefore, proofreading of aaRS is of enormous importance. Discrimination between the right and wrong tRNA is mainly guaranteed by unique recognition elements, like the anticodon or the acceptor stem (Figure 6).[49]
Binding of the correct amino acid is mainly ensured by structural properties and steric hindrance.[50] Some aaRS have a second active site, the so called editing site, responsible for the hydrolysation of tRNAs coupled to wrong amino acids.[51]
Ribosomal decoding
With correctly aminoacylated tRNAs in hand, cells can perform the actual process of protein synthesis, the decoding of mRNA at the ribosome. The ribosome consists of two subunits, a small and a large one, both composed of proteins and ribosomal RNA (rRNA).[50b, 51]
The process of translation can be divided in three phases: initiation, elongation and termination. During initiation, all required factors for translation are assembled, like
Figure 7 Protein translation at the ribosome. During initiation, the functional ribosome is assembled. The polypeptide is formed in the elongation phase. The reaction of peptide bond formation is catalysed by peptidyl transferases. After reaching the stop codon, the process is terminated and the polypeptide is released. Inspired by P. Russell.[4]
18
the mRNA, the small and large ribosomal subunits and the initiator-tRNA. With the functional ribosome, elongation phase of translation can start: Aminoacyl-tRNAs bind to their appropriate codon on the mRNA and a peptidyl transferase catalyses the formation of a peptide bond between two amino acids. This process is promoted by the hydrolysis of GTP to GDP. When reaching a stop codon, the end of the encoded gene, translation is terminated and the generated polypeptide is released (Figure 7).[51]
3.6 Fluorinated amino acids
Fluorine is a unique element with special stereochemical properties, like size, low polarizability and the strongest inductive effect known.[52] Furthermore it is often claimed that fluorine has similar isosteric features as hydrogen. Indeed, the van der Waals radius is only slightly bigger than for hydrogen, whereas the C-F bond is significantly longer than the C-H bond.[53] Fluorine is widely used to modify and optimize pharmaceuticals.[54] Molecules with fluorine moieties can show alteration in hydrophobicity, reactivity, stability or/and conformation.[53, 55] Figure 8 shows all fluorinated amino acids used in this thesis.
The introduction of fluorinated counterparts of natural amino acids in peptides and proteins is widely used to study the impact on polypeptide characteristics.[56] The thermodynamic stabilization of polypeptides by fluorinated variants of aliphatic amino acids is mainly based on increase of hydrophobicity in the hydrophobic core of the polypeptide.[57] As hydrophobicity is also known to be the main factor in protein folding,
Figure 8 Fluorinated amino acids used in this thesis.
Introduction
19
introduction of fluorinated amino acids is used to study and modify this complex mechanism.[57b, 58]
3.7 Engineering and expanding the genetic code
Like fluorinated amino acids, a variety of different non-natural amino acids are used to expand the genetic code. The functional properties of the naturally occurring amino acids are limited and therefore great efforts are done to expand this functionalities to enable researchers for directed manipulation of proteins. Besides the already described possibilities of fluorinated amino acids to stabilize proteins, non-natural amino acids can be used to study biological functions, localization in cells, controlled attachment of complex polymers and many others.[3]
There are several sophisticated methods known, enabling introduction of a brought range of non-natural amino acids in peptides or proteins. For small peptides, solid phase synthesis is a widely used, simply controllable method.[56] For the modification of bigger polypeptides, the natural protein synthesis machinery in cells is the most promising technique. The methods used in this thesis are the “Selective Pressure Incorporation” (SPI) and the “Amber Stop Codon Suppression” (ACS). In the following chapters SPI and ACS will be described in more detail.
Selective Pressure Incorporation (SPI)
SPI bases on two main factors: the auxotrophy of bacterial cells for distinct amino acids and the similarity of the non-natural amino acid to a natural one. The method will be explained on the example of proline, substituted by fluoro proline.
A proline auxotrophic bacterial cell is not able to produce proline on its own, therefore they depend on the addition of proline in the medium. Exchange of the natural proline by the fluorinated counterpart forces the cell to accept the fluorinated proline as substrate and assimilate it.[59]
Transferring this requirements on the experimental procedure, proline auxotrophic cells are transformed with the gene of interest under an inducible promotor and let grown in minimal medium till they consumed all proline in medium. After addition of fluoro proline, expression of the gene of interest is induced and cells incorporate fluoro proline at every position coding for the natural counterpart (Figure 9).
20
As already mentioned, this method has several limitations. The non-natural amino acid has to be very similar to the natural one to be accepted by the endogenous translation machinery, primary by the according aaRS/tRNA pair. The natural amino acid has to be completely consumed before addition of the non-natural counterpart to ensure a total exchange. As this is very difficult to implement in experimental set-ups, some impurities with the natural amino acid cannot be excluded. Finally, if one amino acid is encoded at several positions in a protein, this method leads to the exchange of the natural amino acid by the non-natural amino acid throughout the proteome.
This method is very simple, especially when the experiment requires the protein- or proteome-wide incorporation of the non-natural amino acid or when the amino acid of interest is only encoded once.
Furthermore, some effort was done to increase the incorporation rate of non- natural amino acids. The overexpression of wildtype aaRS can overcome the low affinity of the natural aaRS to the non-natural amino acid substrate.[60] Besides simple overexpression of the aaRS, several groups increased the affinity of the aaRS to the
Figure 9 Selective Pressure Incorporation (SPI). Auxotrophic E.coli cells, transformed with the gene of interest under an inducible promotor are cultured in a minimal medium with limiting concentrations of the amino acid X that has to be exchanged by its non-natural counterpart. After amino acid X is consumed, non-natural amino acid Y is added and the expression of the gene of interest is induced. During translation, the non-natural amino acid Y is incorporated at all naturally occurring positions of amino acid X within the protein.
Introduction
21
non-natural amino acid by evolving the binding pocket, enabling the incorporation of more sterically hindered amino acids.[50b, 61]
Amber Stop Codon Suppression (ACS)
Another method to introduce non-natural amino acids with unique functionalities in proteins is the ACS. In contrast to the former described SPI, this method allows the manipulation of the amino acid sequence at any position, independently from the natural amino acid context.
For this approach, a blank codon is required not coding for a natural amino acid.
As all codons are occupied by natural amino acids, codons have to be “re-purposed”, coding for the non-natural amino acid. Usually, ribosomal protein synthesis is terminated by one of the three stop codons: Amber (UAG), Ochre (UAA) and Opal (UGA). The Amber Stop codon plays a special role, as it is rarely found to terminate essential genes. Therefore, UAG is used as blank codon for non-natural amino acid incorporation. Furthermore, quadruplet codons can be used as blank codons.[62] In this chapter, quadruplet codons will not be further discussed.
As response to a blank codon, the non-natural amino acid has to be incorporated in the nascent peptide chain. Therefore, the non-natural amino acid has to be activated by an aaRS and transferred to a tRNA. The requirements for this, so called, orthogonal aaRS/tRNA pair are the basis for this method: no activation of cognate amino acids and unspecific binding of cognate tRNAs by the orthogonal aaRS, specific recognition of the Amber Stop codon by the orthogonal tRNA and no aminoacylation of the orthogonal tRNA by cognate aaRS. The orthogonal aaRS/tRNA pair is additionally introduced in the expression system, e.g. E.coli, originating from a phylogenetically distant organism. There are several orthogonal pairs frequently used.
The archaea Methanococcus janaschii (now known as Methanocaldococcus) tyrosyl- tRNA synthetase/tRNA pair is orthogonal in E.coli, but not in eukaryotic cells.[3, 63]
Another source is Methanosarcinacea with different pyrrolysyl-tRNA synthetase/tRNA pairs, orthogonal in bacteria, eukaryotic cells and animals.[64] There are also aaRS/tRNA pairs from E.coli, orthogonal in eukaryotic cells.[65] To ensure the specific activation of the non-natural amino acid by the orthogonal aaRS and the correct incorporation in the nascent polypeptide chain by the orthogonal tRNA, in some cases the aaRS/tRNA pair has to undergo evolution. Primary, the active site of the aaRS and the anticodon of the tRNA has to be evolved for the appropriate non-natural amino acid and blank codon (more precisely Amber Stop codon), respectively.[3, 63, 65a] The
22
directed evolution is performed in two steps, first selecting for aaRS from a library activating natural and non-natural amino acids in the presence of the cognate tRNA.
In the second step, aaRS activating natural amino acids are sorted out.[3]
With the evolved orthogonal aaRS/tRNA pair in hand, specific for a non-natural amino acid of interest, the host organism, e.g. E.coli, can be transformed with this genes. Finally, the host cell has to be transformed with the gene of interest, encoding for the protein that has to be modified with the non-natural amino acid. Therefore, the Amber Stop codon is introduced in the gene by site-specific mutagenesis. After induction of gene expression, the non-natural amino acid is incorporated in the protein at a defined position (Figure 10).
3.8 Chemical modification of proteins
Post-translational modifications (PTM) play a significant role in protein structure, function and stability. The high number of different PTM (methylation, acetylation, phosphorylation, ubiquitination, glycosylation, etc.) lead to increasing diversity of the protein pool.[66] Unfortunately, recombinant expression and isolation of site-
Figure 10 Amber Stop codon suppression (ACS). The evolved orthogonal aaRS/tRNA pair activates the non-natural amino acid. The activated non-natural amino acid is incorporated into the nascent polypeptide in response to the Amber Stop codon (UAG). The orthogonal aaRS/tRNA pair does not activate natural amino acids and the orthogonal tRNA is not recognized by endogenous aaRS. Figure inspired by J. Chin.[2b, 3]
Introduction
23
specifically modified proteins for detailed studies of the PTM on protein structure, function or stability is difficult.
To bypass the limitations of isolating specifically modified proteins from living systems, researchers develop a variety of methods to chemically modify proteins with appropriate PTM. These methods can be roughly divided in two fields: modification of natural amino acid residues and the introduction and subsequent modification of non- natural amino acids.[5]
Natural amino acids
Although the functionalities of the naturally occurring amino acids in proteins are limited to alcohols, carboxylic acids, amines, amides and thiols, several methods were developed allowing chemical modifications.
Two natural occurring functionalities play an outstanding role: the thiol group of cysteine and the primary amine of lysine, as well as the N-terminus of the protein.[5]
Cysteine is a good nucleophile, allowing selective reactivity under defined conditions.[66-67] Furthermore, this amino acid is comparatively rare, therefore modification at cysteines can be site-specific for many proteins.[68] A prominent, selective reaction of cysteine residues is performed with maleimides (Michael-type addition, Figure 11A).[69]
Besides cysteine, the primary amine of lysine is another strong nucleophile. But other than cysteine, lysine is a common amino acid in proteins.[70] Therefore, modification of lysine residues is not site-specific and only applicable when modification of all lysine residues is favoured or the site of modification is irrelevant.[71]
To exclude the reaction of thiols, stronger electrophiles have to be used, e.g. activated esters [72] or isothiocyanates.[73]
Reductive alkylation is a further reaction successfully used to modify lysine residues. This reaction is performed in the presence of aldehydes and sodium cyanoborohydride (Figure 11B).[74]
Finally, the N-terminus is a further site for conjugation, showing unique reactivity.
Kent introduced the so called “Native Chemical Ligation” (NCL), allowing the synthetic construction of long peptide backbones. This reaction bases on an N-terminal cysteine residue and a C-terminal thioester resulting in a native peptide bond (Figure 11C).[75]
24 Non-natural amino acids
Although modification at natural amino acids can be applied in several approaches, its limitations are still hindering for many studies. The progress in non-natural amino acid incorporation in proteins, bearing unique chemical functionalities, bypass a lot of this limitations. As already described in the former chapter, non-natural amino acids can be incorporated in proteins i.a. via SPI or ACS. Their unique chemical functionalities can subsequently be used for conjugation of PTM.
Figure 11 Chemical modification of natural amino acid residues and the N-terminus. A Modification of cysteine residues with maleimide (Michael-type addition). B Reductive alkylation of lysine ε-amino group with an aldehyde and sodium cyanoborohydride. C Native Chemical Ligation (NCL) of C-terminal thioester with N-terminal cysteine, resulting in a native peptide bond. Circle: protein, square: protein fragment/ PTM, star: PTM. Inspired by Davis et al.[5]
Figure 12 Chemical modification of non-natural amino acids. A Copper-catalysed azide-alkyne cycloaddition (CuAAC). Azide and alkyne functionalities form a covalent triazole linkage under cooper catalysis. B Strain-promoted azide-alkyne cycloaddition (SPAAC). A cyclooctyne-functionalized protein covalently reacts with an azide. Circle: protein/PTM. Inspired by Davis et al.[5]
Introduction
25
A very popular chemical modification is the copper-catalysed azide-alkyne cycloaddition (CuAAC).[76] As the name already indicates, an azide and an alkyne functionality are required resulting in a triazole linkage (Figure 12A).[77] Both functionalities can be introduced via non-natural amino acids in proteins and then coupled to the appropriate modification bearing the other functionality, e.g. other proteins, PEG chains, glycosylations, etc.[78] Although CuAAC has high specificity and reactivity, copper remains toxic for cells, generating reactive oxygen species.[78b, 79]
In 2004 Bertozzi et al. [80] introduced a copper-free ligation method, the strain- promoted azide-alkyne cycloaddition (SPAAC). This reaction can be performed similarly to the CuAAC, but without the cytotoxic copper catalyst. Like for CuAAC, high specificity and bio-orthogonality was observed (Figure 12B). Furthermore, incorporation of cyclooctynes in proteins via ACS was demonstrated.[81] Nevertheless, synthesis and handling with strained compounds is often challenging.[82]
Another group of coupling methods bases on the Staudinger reaction: the Staudinger ligation.[2a] Again, an azide functionality is required. The second component is a triarylphosphine (Figure 13A).[83] Amongst others, this approach was used to label proteins with fluorophores or photoswitches, showing good biocompatibility.[84] To avoid the residual phosphine oxide moiety, a “traceless”
Staudinger ligation was developed by Raines[85] and Bertozzi[86], resulting in an amide bond (Figure 13B).
Figure 13 Staudinger ligation. A Staudinger ligation between an azide and a triarylphosphine. B Traceless Staudinger ligation, forming an amide bond. Circle: protein, star: PTM. Inspired by Davis et al.[5]
26 Staudinger Phosphite Reaction (SPR)
A coupling reaction playing a crucial role in this thesis is the Staudinger Phosphite Reaction (SPR). Hackenberger et al.[2b] used this reaction for site-specific phosphorylation of proteins. Furthermore, PEGylation could be demonstrated resulting in a branched phosphoramidate-linked PEG-peptide conjugate (Figure 14).[87] The basis for this reaction is again an azide-functionalized protein and a phosphite, e.g. PEG phosphite. The Hackenberger lab established para-azido phenylalanine as azide source.[2b, 87b, 87d, 88] This amino acid can be introduced during solid phase synthesis or via ACS.[56, 89]
Besides its simple mechanism, SPR has a lot of advantages for the project described in this thesis: the site-specific PEGylation of EPO. The reaction is tolerant against denaturing salts and higher temperatures. Coupling can be site-specifically performed in semi-purified protein samples. Furthermore, synthesis of PEG phosphite is simple and cheap and stable for several days at 40°C.[7, 88] As shown in Figure 14, reaction leads to the coupling of branched PEG. This two PEG chains have a positive effect on protein stability as the cause the umbrella effect, shielding a wide range of the protein surface.[40, 88] In contrast to CuAAC, SRP can be performed without toxic supplements (e.g. copper) and under normal atmosphere.[7]
Figure 14 Staudinger Phosphite Reaction (SPR). SPR between an azide-functionalized (para-azido phenylalanine) protein and PEG phosphite, resulting in a site-specifically PEGylated protein via a phosphoramidate linker.[2]
27
28
4. Aim of the Thesis
Biopharmaceuticals are of rising importance to treat a variety of different diseases.
The drawbacks of proteins as pharmaceuticals arises from their instability or even immunogenicity in human bodies. A lot of effort is done to increase protein stability and activity, to decrease frequency of application and cost of production. Currently, this is done by random PEGylation of the amino acids or of the N-terminus, but also via extensive glycosylation and other methods.
The aim of this thesis is to study the impact of non-natural amino acids and site- specific PEGylation on protein stability and activity. EPO, as important biopharmaceutics, is used to perform this studies.
In first approaches, fluorinated amino acids should be incorporated in EPO expressed in E.coli, studying their influence on protein folding and behaviour against increasing temperature and proteases. Afterwards, in vitro assays should demonstrate the preserved receptor binding and activation.
Furthermore, para azido-phenylalanine will be site-specifically incorporated at the naturally occurring glycosylation sites allowing controlled PEGylation of EPO. This experiments should demonstrate the effect of PEGylation on protein behaviour in a position-dependent manner, answering the question if PEG can completely or partially replace the complex glycan chains. Additionally, it will be studied if the applied approaches (ACS and SPR) are potent alternatives for the commonly used methods to PEGylate proteins.
Results and Discussion
29
30
5. Results and Discussion
5.1 Production of EPO and incorporation of fluorinated amino acids
The gene sequence of human EPO was cloned in a pET11a expression vector under the control of the T7 promotor. Furthermore, a C-terminal His6 tag was added to facilitate efficient purification via immobilized metal affinity chromatography (IMAC) (pET11a-WT-EPO). A second construct was accomplished with the gene sequence of the T7 DNA polymerase as some E.coli strains used do not express a T7 RNA polymerase (pET11a-WT-EPO + T7 Pol). This cloning was performed by Martin Hamann.[90] Following amino acid sequence of WT-EPO was used:
MAPPRLICDSR VLERYLLEAK EAENITTGCA EHCSLNENIT VPDTKVNFYA WKRMEVGQQA VEVWQGLALL SEAVLRGQAL LVNSSQPWEP LQLHVDKAVS GLRSLTTLLR ALR114AQKEAIS PPDAASAAPL RTITADTFRK LFRVYSNFLR GKLKLYTGEA CRTGDRHHHH HH
It has to be mentioned that it was worked with an EPO variant, described in literature, showing an exchange of G114 to R114 (UniprotKB-P01588, ENA coding:
CAA26095.1).[10] In the second part of this thesis, it was switched to the endogenous EPO sequence.
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
31
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.
32
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]
Results and Discussion
33
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.
34
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).
