Abstract
This PhD thesis describes the recombinant production of periodic beta-sheet, silk-like polypeptides in Escherichia coli. Polypeptides with the repetitive sequence [(AG)3EG]n (n = 10 - 50; A = alanine, G = glycine and E = glutamic acid) were prepared with a yield of approximately 20 mg per liter of cell culture (n = 20). The polypeptides [(AG)3EG]n (n = 10 and 20) with N- and C-terminal cysteine residues were used for the preparation of hybrid triblock copolymers containing poly(ethylene glycol) (PEG; Mn = 750, 2000 and 5000 g/mol) end blocks. In the solid state, the polypeptide block is known to adopt a regular antiparallel beta-sheet conformation with glutamic acid residues at the turn positions. This conformation was retained upon conjugation with the various PEGs as was indicated by infra-red spectroscopy. Whereas the polypeptide alone formed large aggregates, transmission electron microscopy (TEM) analysis showed well-defined fibrillar structures with a width of approximately 12 nm for [(AG)3EG]20-PEG750 triblock copolymer. Based on TEM and atomic force microscopy microscopy (AFM) data fibril formation is proposed to occur in the beta-sheet stacking direction. The presence of reactive groups at the fibril surface could be useful, for example, as a scaffold in the generation of conducting nanowires. Furthermore, the design of an alpha-helical polypeptide is presented, which could act as a scaffold for the preparation of well-defined glycopolymers. A designed polypeptide with the repetitive sequence [(MAKA)2MAA]n (M = methionine) harbours methionine and lysine residues at opposing sides of the helical axis. Whereas lysine residues can be directly used for chemical functionalization, replacement of methionine with the non-proteinogenic analogue 2-amino-5-hexynoic acid, should provide a handle for an efficient conjugation via [3+2]-cycloaddition. Although circular dichroism analysis of small peptides showed that the designed sequence was prone to adopt an alpha-helical conformation, the recombinant production of longer polypeptides in Escherichia coli was unsuccessful.
