The preparation of genetically engineered protein-based polymers, where the polypeptides can be prepared with precise control of the sequence, molecular weight and stereochemistry, is an exciting and very promising area of polymer chemistry. A unique aspect of genetically engineered polymeric materials is that the preparation of the biochemical polymer template, synthesis of the coding DNA sequence, is required only once. Subsequent biosynthetic processes obviate the need for difficult or complex synthetic manipulations. In particular, modern cloning techniques facilitate the systematic substitution of unique or regular sites in very large polymers.
Rational design of polypeptide-based polymers, that fold via established rubrics to form specific secondary structures, enables the programming of those mechanical or physical properties1,2 into the sequence that may be desirable for drug delivery or protein purification. The construction of β-sheet forming polypeptides with utility as constructs for the study of β-sheet folding, aggregation or amyloid formation is possible. Ultimately the self-assembly of such molecular building blocks may also find applications in modern nanotechnology where precise dimensional control and selective functionalization are crucial
The design and rapid construction of libraries of genes coding β-sheet forming repetitive and block-copolymerized polypeptides bearing various C- and N-terminal sequences was based on the assembly of DNA cassettes coding for the (GA)3GX amino acid sequence where the (GAGAGA) sequences would constitute the β-strand units of a larger β-sheet assembly. The edges of this β-sheet would be functionalized by the turn-inducing amino acids (GX). The polypeptides were expressed in E. coli using conventional vectors and were purified by Ni-NTA chromatography
A de novo, genetically engineered 687 residue polypeptide consisting of repetitive polypeptides with 32 amino acid repeats, (GA)3GY(GA)3GE(GA)3GH(GA)3GK (32YEHK) expressed in E. coli has been found to form highly rectilinear, β-sheet containing fibrillar structures. Tapping-mode atomic force microscopy, deep-UV Raman spectroscopy and transmission electron microscopy definitively established the tendency of the fibrils to predominantly display an apparently planar bilayer or ribbon assemblage.
The de novo polypeptide GH6[(GA)3GY(GA)3GE]8GAH6 (YE8) was designed and genetically engineered to form antiparallel β-strands of GAGAGA repeats. Modulation of pH enables control of solubility, folding and aggregation of YE8 by control of the overall polypeptide charge, a consequence of the protonation or deprotonation of the glutamic acid and histidine residues. YE8 exhibits all the major properties of a fibrillogenic protein providing an excellent model for detailed study of the fibrillation. At neutral pH, YE8 is soluble in disordered form, yet at pH 3.5 folds into a predominantly β-sheet conformation that is fibrillogenic. Atomic force microscopy and transmission electron microscopy indicated the formation of fibrilar aggregates on well-defined, hydrophobic surfaces. The β-sheet folding of YE8 exhibited a lag phase that could be eliminated by seeding or stirring. The strong dependence of lag time on polypeptide concentration established the limiting step in aggregation as initiation of β-sheet folding.