Developing functionality in elastin-like polymers by increasing their molecular complexity: the power of the genetic engineering approach (original) (raw)
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Genetic engineering of structural protein polymers
Biotechnology Progress
Genetic and protein engineering are components of a new polymer chemistry that provide the tools for producing macromolecular polyamide copolymers of diversity and precision far beyond the current capabilities of synthetic polymer chemistry. The genetic machinery allows molecular control of chemical and physical chain properties. Nature utilizes this control to formulate protein polymers into materials with extraordinary mechanical properties, such as the strength and toughness of silk and the elasticity and resilience of mammalian elastin. The properties of these materials have been attributed to the presence of short repeating oligopeptide sequences contained in the proteins, fibroin, and elastin. We have produced homoblock protein polymers consisting exclusively of silk-like crystalline blocks and elastin-like flexible blocks. We have demonstrated that each homoblock polymer as produced by microbial fermentation exhibits measurable properties of crystallinity and elasticity. Additionally, we have produced alternating block copolymers of various amounts of silk-like and elastin-like blocks, ranging from a ratio of 1:4 to 2:1, respectively. The crystallinity of each copolymer varies with the amount of crystalline block interruptions. The production of fiber materials with custom-engineered mechanical properties is a potential outcome of this technology.
Elastin-like polypeptides as a promising family of genetically-engineered protein based polymers
World Journal of Microbiology and Biotechnology, 2014
Elastin-like polypeptides (ELP) are artificial, genetically encodable biopolymers, belonging to elastomeric proteins, which are widespread in a wide range of living organisms. They are composed of a repeating pentapeptide sequence Val-Pro-Gly-Xaa-Gly, where the guest residue (Xaa) can be any naturally occurring amino acid except proline. These polymers undergo reversible phase transition that can be triggered by various environmental stimuli, such as temperature, pH or ionic strength. This behavior depends greatly on the molecular weight, concentration of ELP in the solution and composition of the amino acids constituting ELPs. At a temperature below the inverse transition temperature (T t), ELPs are soluble, but insoluble when the temperature exceeds T t. Furthermore, this feature is retained even when ELP is fused to the protein of interest. These unique properties make ELP very useful for a wide variety of biomedical applications (e.g. protein purification, drug delivery etc.) and it can be expected that smart biopolymers will play a significant role in the development of most new materials and technologies. Here we present the structure and properties of thermally responsive elastin-like polypeptides with a particular emphasis on biomedical and biotechnological application.
Nanobiotechnological approach to engineered biomaterial design: the example of elastin-like polymers
Nanomedicine, 2006
Today, the development of advanced biomaterials is still lacking an appropriate tailored engineering approach. Most of the biomaterials currently used have their origin in materials developed for other technological applications. This lack of adequate biomaterial design is probably due to the peculiar environment where those materials must operate. On the one hand, this environment is dominated by the immune rejection system. On the other hand, the functionality of natural biomolecules is based on complex topological physical–chemical function distributions at the nanometer level. This review presents arguments concerning the role of biotechnology and nanotechnology in the future development of new advanced biomaterials and the potential of these biomaterials as a way to achieve highly biofunctional and truly biocompatible biomaterials for hot areas, such as regenerative medicine and controlled release. Recombinant protein–polymers will be presented as an example of candidates for t...
Protein-based materials, toward a new level of structural control
Chem. Commun., 2001
Through billions of years of evolution nature has created and refined structural proteins for a wide variety of specific purposes. Amino acid sequences and their associated folding patterns combine to create elastic, rigid or tough materials. In many respects, nature's intricately designed products provide challenging examples for materials scientists, but translation of natural structural concepts into bio-inspired materials requires a level of control of macromolecular architecture far higher than that afforded by conventional polymerization processes. An increasingly important approach to this problem has been to use biological systems for production of materials. Through protein engineering, artificial genes can be developed that encode protein-based materials with desired features. Structural elements found in nature, such as b-sheets and a-helices, can be combined with great flexibility, and can be outfitted with functional elements such as cell binding sites or enzymatic domains. The possibility of incorporating non-natural amino acids increases the versatility of protein engineering still This journal is
Synthetic biology through biomolecular design and engineering
Current Opinion in Structural Biology, 2008
We describe a straightforward single-peptide design that self-assembles into extended and thickened nano-to-mesoscale fibers of remarkable stability and order. The basic chassis of the design is the well-understood dimeric R-helical coiled-coil motif. As such, the peptide has a heptad sequence repeat, abcdefg, with isoleucine and leucine residues at the a and d sites to ensure dimerization. In addition, to direct staggered assembly of peptides and to foster fibrillogenesissthat is, as opposed to blunt-ended discrete speciessthe terminal quarters of the peptide are cationic and the central half anionic with lysine and glutamate, respectively, at core-flanking e and g positions. This +,-,-,+ arrangement gives the peptide its name, MagicWand (MW). As judged by circular dichroism (CD) spectra, MW assembles to R-helical structures in the sub-micromolar range and above. The thermal unfolding of MW is reversible with a melting temperature >70°C at 100 µM peptide concentration. Negative-stain transmission electron microscopy (TEM) of MW assemblies reveals stiff, straight, fibrous rods that extended for tens of microns. Moreover, different stains highlight considerable order both perpendicular and parallel to the fiber long axis. The dimensions of these features are consistent with bundles of long, straight coiled R-helical coiled coils with their axes aligned parallel to the long axis of the fibers. The fiber thickening indicates intercoiled-coil interactions. Mutagenesis of the outer surface of the peptidesi.e., at the b and f positionsscombined with stability and microscopy measurements, highlights the role of electrostatic and cation-π interactions in driving fiber formation, stability and thickening. These findings are discussed in the context of the growing number of self-assembling peptide-based fibrous systems. † This work was funded by the BBSRC of the U.K.
Robust high-throughput synthesis methods are needed to expand the repertoire of repetitive protein-polymers for different applications. To address this need, we developed a new method, overlap extension rolling circle amplification (OERCA), for the highly parallel synthesis of genes encoding repetitive protein-polymers. OERCA involves a single PCR-type reaction for the rolling circle amplification of a circular DNA template and simultaneous overlap extension by thermal cycling. We characterized the variables that control OERCA and demonstrated its superiority over existing methods, its robustness, high-throughput and versatility by synthesizing variants of elastin-like polypeptides (ELPs) and protease-responsive polymers of glucagon-like peptide-1 analogues. Despite the GC-rich, highly repetitive sequences of ELPs, we synthesized remarkably large genes without recursive ligation. OERCA also enabled us to discover 'smart' biopolymers that exhibit fully reversible thermally responsive behaviour. This powerful strategy generates libraries of repetitive genes over a wide and tunable range of molecular weights in a 'one-pot' parallel format. A rtificial repetitive polypeptides-also termed protein-polymers-derived from short peptide motifs found in elastin, collagen, silk and other structural proteins exhibit unique mechanical, structural and biological properties. These attributes have led to their application in biotechnology, tissue engineering, drug delivery and biosensing 1-5. Recombinant DNA technology is attractive for the synthesis of protein-polymers because it enables precise control of their length (number of repeats), composition and stereochemistry. This level of control is especially important for the in vivo applications of these biopolymers, because the polymer molecular weight controls their pharmacokinetics and biodistribution, whereas the amino acid sequence imparts biological activity to the biopolymer and affects their route, rate and mechanism of biodegradation. Recombinant DNA technology is also of interest for the synthesis of tandem repeats of naturally occurring peptides, as a strategy for the high-yield synthesis of peptide drugs and antigens 6-10. Furthermore, polymerization of peptide drugs with intervening protease cleavable sequences has the potential to improve their pharmacokinetics and drug efficacy 11,12. However, current methods for the polymerization of DNA suffer from one or more critical limitations: they (1) require numerous steps, (2) are difficult to run in parallel, and (3) do not provide tunable control over a range of molecular weights, all of which greatly limit the ability to simultaneously synthesize multiple variants with a range of repeat units and compositions. Motivated by these limitations, we report a rapid, one-step, high-throughput and robust method for the recombinant polymerization of 'monomer' DNA sequences with tunable control over the number of repeats. This method, which we term overlap extension rolling circle amplification (OERCA), uses rolling circle amplification (RCA) to produce linear repeats of a circularized gene, followed by thermally cycled overlap extension (OE) to yield a library of polymers of the monomer DNA, all in a single PCR reaction. Here we show the utility of OERCA and its advantages over existing methods by the synthesis of two classes of protein-polymers. First, we demonstrate the parallel synthesis of genes that encode elastin-like polypeptides (ELPs), a family of thermally responsive protein-polymers derived from a recurring VPGVG pentapeptide found in elastin 13. We used OERCA to rapidly synthesize nine different variants of ELPs, by substituting or inserting alanine residues along the VPGVG motif. These studies revealed an unexpected degree of sequence promiscuity in the parent peptide motif and yielded new families of 'smart' protein-polymers that exhibit fully reversible thermally responsive behaviour, which will provide a new set of stimulus responsive motifs for biomedical and biotechnological applications. In a second example, we use OERCA to rapidly synthesize protease-responsive polymers of glucagon-like peptide-1 (GLP-1) analogues, with intervening protease sites of variable potency for the optimization of in vivo pharmacokinetics and the release of GLP-1 from the polymer.
We report a new strategy for the synthesis of genes encoding repetitive, protein-based polymers of specified sequence, chain length, and architecture. In this stepwise approach, which we term "recursive directional ligation" (RDL), short gene segments are seamlessly combined in tandem using recombinant DNA techniques. The resulting larger genes can then be recursively combined until a gene of a desired length is obtained. This approach is modular and can be used to combine genes encoding different polypeptide sequences. We used this method to synthesize three different libraries of elastin-like polypeptides (ELPs); each library encodes a unique ELP sequence with systematically varied molecular weights. We also combined two of these sequences to produce a block copolymer. Because the thermal properties of ELPs depend on their sequence and chain length, the synthesis of these polypeptides provides an example of the importance of precise control over these parameters that is afforded by RDL.
Genetically Engineered Elastin-based Biomaterials for Biomedical Applications
Current medicinal chemistry, 2018
Protein-based polymers are some of the most promising candidates for a new generation of innovative biomaterials as recent advances in genetic-engineering and biotechnological techniques mean that protein-based biomaterials can be designed and constructed with a high degree of complexity and accuracy. Moreover, their sequences, which are derived from structural protein-based modules, can easily be modified to include bioactive motifs that improve their functions and material-host interactions, thereby satisfying fundamental biological requirements. The accuracy with which these advanced polypeptides can be produced, and their versatility, self-assembly behavior, stimuli-responsiveness and biocompatibility, means that they have attracted increasing attention for use in biomedical applications such as cell culture, tissue engineering, protein purification, surface engineering and controlled drug delivery. The biopolymers discussed in this review are elastin-derived protein-based polym...
How to create polymers with protein-like capabilities: A theoretical suggestion
Physica D: Nonlinear Phenomena, 1997
The synthesis of a man-made polymer capable of functioning in a protein-like fashion can be of tremendous technological importance, dnd may also shed light on the natural creation of the molecular basis of life. In case of proteins, the unique 3D fold, responsible for the particular functionality of the molecule, is determined by the particular sequence of monomer units. We suggest a procedure, which we call imprinting, to control the monomer sequence of an artificial heteropolymer during its synthesis in order to obtain a heteropolymer with the protein-like properties of quick and reliable renaturability to some unique spatial fold capable of certain functional properties. To control the sequence formation, our procedure employs interactions between monomers. We will show that this leads to renaturable chains, because renaturation is governed exactly by the same interactions between monomer units. We present here both analytical and computational study of imprinting, yielding the requirements on the set of monomers chosen and further more specific prescriptions for the experimental verification of this theory.