Refine
Document Type
- Doctoral Thesis (4) (remove)
Is part of the Bibliography
- yes (4)
Keywords
- polypeptide (4) (remove)
Institute
Plants are an attractive platform for the production of medicinal compounds because of their potential to generate large amounts of biomass cheaply. The use of chloroplast transformation is an attractive way to achieve the recombinant production of proteins in plants, because of the chloroplasts’ high capacity to produce foreign proteins in comparison to nuclear transformed plants. In this thesis, the production of two different types of antimicrobial polypeptides in chloroplasts is explored.
The first example is the production of the potent HIV entry inhibitor griffithsin. Griffithsin has the potential to prevent HIV infections by blocking the entry of the virus into human cells. Here the use of transplastomic plants as an inexpensive production method for griffithsin was explored. Transplastomic plants grew healthily and were able to accumulate griffithsin to up to 5% of the total soluble protein. Griffithsin could easily be purified from tobacco leaf tissue and had a similarly high neutralization activity as griffithsin recombinantly produced in bacteria. Griffithsin could be purified from dried tobacco leaves, demonstrating that dried leaves could be used as a storable starting material for griffithsin purification, circumventing the need for immediate purification after harvest.
The second example is the production of antimicrobial peptides (AMPs) that have the capacity to kill bacteria and are an attractive alternative to currently used antibiotics that are increasingly becoming ineffective. The production of antimicrobial peptides was considerably more challenging than the production of griffithsin. Small AMPs are prone to degradation in plastids. This problem was overcome by fusing AMPs to generate larger polypeptides. In one approach, AMPs were fused to each other to increase size and combine the mode of action of multiple AMPs. This improved the accumulation of AMPs but also resulted in impaired plant growth. This was solved by the use of two different inducible systems, which could largely restore plant growth. Fusions of multiple AMPs were insoluble and could not be purified.
In addition to fusing AMPs to each other, the fusion of AMPs to small ubiquitin-like modifier (SUMO), was tested as an approach to improve the accumulation, facilitate purification, and reduce the toxicity of AMPs to chloroplasts. Fusion of AMPs to SUMO indeed increased accumulation while reducing the toxicity to the plants. SUMO fusions produced inside chloroplasts could be purified, and SUMO could be efficiently cleaved off with the SUMO protease. Such fusions therefore provide a promising strategy for the production of AMPs and other small polypeptides inside chloroplasts.
Proteins are natural polypeptides produced by cells; they can be found in both animals and plants, and possess a variety of functions. One of these functions is to provide structural support to the surrounding cells and tissues. For example, collagen (which is found in skin, cartilage, tendons and bones) and keratin (which is found in hair and nails) are structural proteins. When a tissue is damaged, however, the supporting matrix formed by structural proteins cannot always spontaneously regenerate. Tailor-made synthetic polypeptides can be used to help heal and restore tissue formation.
Synthetic polypeptides are typically synthesized by the so-called ring opening polymerization (ROP) of α-amino acid N-carboxyanhydrides (NCA). Such synthetic polypeptides are generally non-sequence-controlled and thus less complex than proteins. As such, synthetic polypeptides are rarely as efficient as proteins in their ability to self-assemble and form hierarchical or structural supramolecular assemblies in water, and thus, often require rational designing. In this doctoral work, two types of amino acids, γ-benzyl-L/D-glutamate (BLG / BDG) and allylglycine (AG), were selected to synthesize a series of (co)polypeptides of different compositions and molar masses.
A new and versatile synthetic route to prepare polypeptides was developed, and its mechanism and kinetics were investigated. The polypeptide properties were thoroughly studied and new materials were developed from them. In particular, these polypeptides were able to aggregate (or self-assemble) in solution into microscopic fibres, very similar to those formed by collagen. By doing so, they formed robust physical networks and organogels which could be processed into high water-content, pH-responsive hydrogels. Particles with highly regular and chiral spiral morphologies were also obtained by emulsifying these polypeptides. Such polypeptides and the materials derived from them are, therefore, promising candidates for biomedical applications.
Es wurden Poly(2-isopropyl-2-oxazolin)-Makroinitiatoren mit terminaler Ammoniumtrifluoracetat-Endgruppe synthetisiert, die anschließend für die Ammonium vermittelte NCA Polymerisation in NMP eingesetzt wurden. Die hierbei synthetisierten Poly(2-isopropyl-2-oxazolin)-block-poly(L-glutamat) (PIPOX-PEP) Blockcopolymere hatten eine Molekulargewichtsverteilung von 1,2 (UZ). Es wurde beobachtet, dass Poly(2-isopropyl-2-oxazolin) bei langen Zeiten oberhalb der LCST irreversibel sphärische Strukturen bildet, die eine hierarchische Struktur besitzen und bei denen es sich möglicherweise um "large compound micelles" handelt. PIPOX-PEP kann in wässeriger Lösung bei langen Zeiten oberhalb der LCST "cottonball" Strukturen bilden. Die Aggregate wurden mittels Lichtstreuung, NMR und TEM charakterisiert. Im Rahmen der Arbeit wurden Strukturbildungsmodelle entwickelt.
Die vorliegende Arbeit beschäftigt sich mit der Synthese und den Eigenschaften von linearen und verzweigten amphiphilen Polypeptid-Blockcopolymeren. Die Frage nach dem Einfluss der Topologie und Konformation der Blockcopolymere auf die supramolekularen und kolloidalen Eigenschaften bildete einen wichtigen Aspekt bei den Untersuchungen. Die Blockcopolymere wurden nach einem mehrstufigen Reaktionsschema durch Kombination von anionischer und ringöffnender Polymerisation von Aminosäuren-N-Carboxyanhydriden (NCA) synthetisiert. Die Untersuchung der Polypeptid-Blockcopolymere hinsichtlich ihres Aggregationsverhaltens in fester Phase sowie in verdünnter wässriger Lösung erfolgte mittels Streumethoden (SAXS, WAXS, DLS) sowie abbildender Methoden (TEM). Durch Einsatz der Blockcopolymere als polymere Stabilisatoren in der Emulsionspolymerisation wurden Oberflächen funktionalisierte Latizes erhalten. Als Beispiel für eine pharmazeutische Anwendung wurden bioverträgliche Polypeptid-Blockcopolymere als Wirkstoff-Trägersysteme in der Krebstherapie eingesetzt.