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in Vivo
Published in Harishkumar Madhyastha, Durgesh Nandini Chauhan, Nanopharmaceuticals in Regenerative Medicine, 2022
Katie M. Kilgour, Brendan L. Turner, Augustus Adams, Stefano Menegatti, Michael A. Daniele
Finally, synthetic peptides represent special building blocks for tissue scaffold engineering, being biocompatible, biodegradable, bioactive, and low-toxicity protein mimetics (Du, Zhou, Shi, & Xu, 2015; Liu, Zhang, Zhu, Liu, & Chen, 2019). Peptides can assemble into hydrogels either physically, by forming a network of hydrogen bonds and electrostatic and hydrophobic interactions, or chemically, via site-selective crosslinking (Liu et al., 2019). Lastly, they can be manufactured in high volumes, affordably, and with no variability. This makes peptides ideal biomaterials for tissue engineering and wound healing (Barbosa & Martins, 2017). In this context, elastin-like polypeptides are of particular interest, as they feature a thermo-responsive phase behaviour, wherein the sol–gel transition temperature and the mechanical properties of the resulting hydrogel depend on the ELP’s amino acid sequence (Urry, 1997). Engineered ELPs fused with cell-binding peptide domains or signalling proteins have been expressed and utilised as building blocks to construct different angiogenic substrates (e.g., gels, films, or fibres) with tuned ELP: water ratio, temperature, and composition of the growth medium. Crosslinked ELP hydrogels have been constructed to present cell-binding motifs such as fibronectin-derived REDV, VEGF-derived QK, laminin-derived IKVAV, and integrin-binding RGD to act as ECM mimetics. Cai et al. developed ELP-based hydrogels encapsulating HUVECs and functionalised with the cell-binding RGD sequence and the VEGF mimetic QK peptide; the hydrogels maintained nearly 100% of cell viability along with significantly enhanced cell proliferation and 3D outgrowth (Cai, Dinh, & Heilshorn, 2014). Another study, by Santos et al., focused on cell- and factor-free hybrid hydrogels constructed with ELPs, polyethylene glycol (PEG), and the self-assembling IKVAV peptide and evaluated their ability to induce angiogenesis and innervation in vivo (dos Santos et al., 2019); notably, the hydrogel hosted a larger density of vessels 26 days post-implantation when the IKVAV peptide was present, indicating that integration of bioactive peptides in natural biomaterials provides a route towards long-term stability in pro-angiogenic scaffolds.
Designing an ELP-intein system: toward a more realistic outlook
Published in Preparative Biochemistry and Biotechnology, 2019
Saeed Ranjbar, Fatemeh Rahbarizadeh, Davoud Ahmadvand
Recombinant proteins are now essential items in contemporary medicine, with a wide range of therapeutic, diagnostic, and industrial applications.[1] The exponential growth of the recombinant protein market in recent years necessitates the emergence of more simple and economical methods of protein purification as the most challenging section of protein production.[2] Introduction of affinity tags in the early 1980s had an undeniable impact on the development of recombinant technology, based on which typically fusion tags (e.g. 6X His, MBD, CBD) is fused to the target protein at DNA level, enabling their one-step purification using their specific affinity to a particular ligand (e.g. metal ions, maltose, cellulose) fixed on the column.[3] Despite being widely employed, the effectiveness of this system has been limited, in part, due to the well-known deficiencies associated with chromatography and elimination of the affinity tags. Limited loadable protein content supplied by ligand presenting chromatography resins,[2] gradual separation of the ligands from affinity resins and attenuation of the final product during several elution steps,[4] along with being a relatively time consuming and expensive technique,[5] have noticeably confined the utilization of affinity tagging in an industrial scale. This has compelled scientists to seek for alternative methods of bioseparation, which led to the advent of aggregation tags.[6] Elastin-like polypeptides (ELPs) are a major class of aggregation tags, which can be employed to purify fused proteins non-chromatographically, through heating and centrifugation. Being composed of repeats of the pentapeptide (Val-Pro-Gly-Xaa-Gly)n where Xaa can be any amino acid except Proline,[7] ELPs undergo a sharp and reversible phase transition at a specific temperature known as the inverse transition temperature (Tt). In a reversible manner, below its Tt, an ELP is highly soluble in aqueous solution whereas if the solution is heated and Tt is reached, ELPs becomes insoluble and can be easily separated by centrifugation [8] or ultrafiltration.[9] The ELP genes of different lengths are typically synthesized by a process called recursive directional ligation throughout which short gene segments are seamlessly combined in tandem using recombinant DNA techniques to build a specified ELP polymer.[10]