Bioproduction and self-assembly of designer peptides

Abstract

Self-assembly is a powerful method for producing controlled morphologies at the nanometre scale. Peptides are biomolecules capable of self-assembly and have the potential for use in a variety of applications such as emulsion and foam stabilisation, wound healing and drug delivery. The investigation of peptide sequence-structure relationships is a rapidly advancing field of study, which in many cases now allows the targeted design of self-assembling peptides. While self-assembling peptides show potential in several fields, their large-scale adoption is limited by the high production expenses associated with conventional solid-phase synthesis. A potentially cheaper and more renewable approach to peptide production is bacterial expression. However, the bioproduction of peptides is a non-trivial process, and generally involves the expression of peptides as part of a fusion protein in which the target peptide is only a small portion of the expressed product. An alternate approach involves peptide concatemers, in which the target peptide makes up the majority of the expressed construct. The work reported in this thesis focused on the design, characterisation and bioproduction of self-assembling a-helical peptides. It was particularly focussed on the development of amphiphillic a-helical peptides with applications as surfactants and hydrogelators. The aim of this work was to further the field of de novo peptide design, while also investigating an approach for peptide bioproduction. This work aimed to combine the requirements for peptide bioproduction with end-use functionality. Chapter 2 details successful bioproduction of an anionic helical surfactant peptide EDP-11, as part of a charge-paired heteroconcatemer with the cationic expression partner RDP-4. The method utilised designed assembly of the constituent a-helical peptides to generate a stably folded coiled-coil concatemer. The polypeptide sequence was further optimized for molecular charge, hydropathy and predicted protease resistance. This process allowed expression of a soluble concatemer that accumulated to high levels (22% of total protein) in E. coli. The expressed concatemer possessed extreme stability to heat and proteases, allowing isolation by simple heat and pH precipitation, yielding concatemer at 130 mg per gram of dry cell weight and g99% purity. Key parameters used in designing the heteroconcatemer were then compared to those of all open reading frames of several reference proteomes in an attempt to gain insight into the mechanistic basis for the high stability of the designed miniprotein. Following bioproduction using this concatemer approach, further processing was required to produce monomeric peptides for use in surfactant applications. The design of acid-cleavable aspartate-proline sites within the concatemer sequence allowed for simple heat- and acid-mediated cleavage to give constituent peptides. Chapter 3 details characterization of cleavage of the expressed concatemer by coupled liquid chromatography-mass spectrometry and includes modelling of the kinetic pathways involved. Chemical denaturation studies showed that cleavage decreased the stability of the coiled coil from 38.9 to 32.8 kcal mol-1. Both intact and cleaved concatemer possessed surfactant functionality, with each giving an equilibrium interfacial pressure of 29 mN m-1 at the air-water interface. Both concatemer cleavage and addition of guanidinium chloride to partially denature coiled-coil structure resulted in enhanced rates of adsorption to the interface. The cleaved products were also used to prepare heat-stable oil-in-water emulsions with droplet sizes in the nanometre range. Chapter 4 describes the design and characterisation of peptide AFD19, which was designed as part of ongoing work on amphiphilic a-helical peptides. AFD19 self-assembled to form fibrils and hydrogels at weight fractions below 0.1%. Gelation occurred in a pH-dependent manner, which was attributed to changes in molecular charge. AFD19 gave free-flowing solutions at high molecular charge, gel formation where the peptide charge was close to p1, and precipitation when the charge approached zero. Characterisation of AFD19 self-assembly indicated the peptide assembled to give coiled-coil fibrils of approximately a hexamer in cross-section that formed physical cross-links below a critical molecular charge. While AFD19 precipitated at close to neutral pH, redesign of the peptide sequence gave AFD36, which underwent gelation at physiological pH and salt, increasing utility in the biomaterial field. Small-angle X-ray scattering showed AFD36 to form fibrils of 3.8-3.9 nm diameter at pH 4.0-7.0. Coiled coils of both peptides possessed high thermodynamic stability, with DG(H2O) values of 9.3-9.5 kcal mol-1 per monomer. Chapter 5 details the investigation of a-helical peptide hydrogels as cell scaffolds and therapeutic delivery vehicles. Rheological measurements of an AFD36 hydrogel showed viscoelastic properties with an elastic modulus of 350 Pa. Further peptide design gave the sequence AFD49, and hydrogels formed by either this peptide or AFD36 supported the growth of NIH/3T3 fibroblasts at levels similar to controls of tissue culture polystyrene. AFD49 hydrogels also supported the proliferation of encapsulated fibroblasts over several weeks, but were unable to support the growth of induced pluripotent stem cells in the absence of coating with Matrigel. The ability of AFD49 hydrogels to incorporate and release the hydrophobic drug all-trans retinoic acid was also demonstrated.