Design of a single-chain polypeptide tetrahedron assembled from coiled-coil segments.

Protein structures evolved through a complex interplay of cooperative interactions, and it is still very challenging to design new protein folds de novo. Here we present a strategy to design self-assembling polypeptide nanostructured polyhedra based on modularization using orthogonal dimerizing segments. We designed and experimentally demonstrated the formation of the tetrahedron that self-assembles from a single polypeptide chain comprising 12 concatenated coiled coil-forming segments separated by flexible peptide hinges. The path of the polypeptide chain is guided by a defined order of segments that traverse each of the six edges of the tetrahedron exactly twice, forming coiled-coil dimers with their corresponding partners. The coincidence of the polypeptide termini in the same vertex is demonstrated by reconstituting a split fluorescent protein in the polypeptide with the correct tetrahedral topology. Polypeptides with a deleted or scrambled segment order fail to self-assemble correctly. This design platform provides a foundation for constructing new topological polypeptide folds based on the set of orthogonal interacting polypeptide segments.

Functional self-assembling polypeptide bionanomaterials.

Bionanotechnology seeks to modify and design new biopolymers and their applications and uses biological systems as cell factories for the production of nanomaterials. Molecular self-assembly as the main organizing principle of biological systems is also the driving force for the assembly of artificial bionanomaterials. Protein domains and peptides are particularly attractive as building blocks because of their ability to form complex three-dimensional assemblies from a combination of at least two oligomerization domains that have the oligomerization state of at least two and three respectively. In the present paper, we review the application of polypeptide-based material for the formation of material with nanometre-scale pores that can be used for the separation. Use of antiparallel coiled-coil dimerization domains introduces the possibility of modulation of pore size and chemical properties. Assembly or disassembly of bionanomaterials can be regulated by an external signal as demonstrated by the coumermycin-induced dimerization of the gyrase B domain which triggers the formation of polypeptide assembly.

Characterization of membrane adsorbers used for impurity removal during the continuous purification of monoclonal antibodies.

A fully continuous, downstream process represents one of the most interesting novel purification approaches in the biosimilars industry, because it would enhance the production output while reducing the costs of complex biopharmaceuticals. Since it generally involves several chromatographic steps, the selection of appropriate chromatographic columns is of utmost importance. In this study we compared several commercially available ion-exchange-membrane adsorbers (NatriFlo®, Sartobind® and Mustang®) for the removal of deoxyribonucleic acid (DNA), host cell proteins (HCPs) and monoclonal antibody aggregates in flow-through mode. Design of Experiments (DoEs) was employed to determine the optimal pH and conductivity conditions. We demonstrated that all the anion-exchange-membrane adsorbers were capable of removing DNA and HCPs from monoclonal antibody mixtures below the required threshold across a wide range of sample pH and conductivity values, and that the HCPs' normalized outlet concentration increases almost linearly with the loading, being independent of the HCPs' concentration. No significant differences in the profile of the adsorbed HCPs with respect to the membrane adsorbers were observed, based on 2D electrophoresis analysis data, although they exhibited different binding capacities. Cation-exchange-membrane adsorbers were also tested for the removal of aggregates. The Yamamoto model was used to determine the number of binding sites and estimate the conductivity range for efficient removal of aggregates, while maintaining a high monoclonal antibody recovery. However, the obtained range had to be further fine-tuned experimentally, due to displacement phenomena. Differences in the trends of binding-site number with a change in the pH value for the tested cation-exchange adsorbers indicate slightly different adsorption mechanisms. To obtain optimal process performance, adjustments to the pH and the conductivity were required between the anion- and cation-exchange steps.

TOPOFOLD, the designed modular biomolecular folds: polypeptide-based molecular origami nanostructures following the footsteps of DNA.

Biopolymers, the essential components of life, are able to form many complex nanostructures, and proteins in particular are the material of choice for most cellular processes. Owing to numerous cooperative interactions, rational design of new protein folds remains extremely challenging. An alternative strategy is to design topofolds-nanostructures built from polypeptide arrays of interacting modules that define their topology. Over the course of the last several decades DNA has successfully been repurposed from its native role of information storage to a smart nanomaterial used for nanostructure self-assembly of almost any shape, which is largely because of its programmable nature. Unfortunately, polypeptides do not possess the straightforward complementarity as do nucleic acids. However, a modular approach can nevertheless be used to assemble polypeptide nanostructures, as was recently demonstrated on a single-chain polypeptide tetrahedron. This review focuses on the current state-of-the-art in the field of topological polypeptide folds. It starts with a brief overview of the field of structural DNA and RNA nanotechnology, from which it draws parallels and possible directions of development for the emerging field of polypeptide-based nanotechnology. The principles of topofold strategy and unique properties of such polypeptide nanostructures in comparison to native protein folds are discussed. Reasons for the apparent absence of such folds in nature are also examined. Physicochemical versatility of amino acid residues and cost-effective production makes polypeptides an attractive platform for designed functional bionanomaterials.

New designed protein assemblies.

Self-assembly is an essential concept of all organisms. Polypeptides self-assemble either within a single polypeptide chain or through assembly of protein domains. Recent advances in designed protein assemblies were achieved by genetic or chemical linkage of oligomerization domains and by engineering new interaction interfaces, which resulted in formation of lattices and cage-like protein assemblies. The absence of new experimentally determined protein folds in the last few years underlines the challenge of designing new folds. Recently a new strategy for designing self-assembly of a polypeptide fold, based on the topological arrangement of coiled-coil modules as the protein origami, has been proposed. The polypeptide tetrahedron was designed from a single chain concatenating of coiled-coil forming building modules interspersed with flexible hinges. In this strategy the order of coiled-coil segments defines the fold of the polypeptide nanostructure.