Elastin-like peptides (ELPs) are derivatives of tropoelastin with a unique property that allows them to stay soluble below a certain critical temperature but reversibly form aggregates above that temperature. Since they are derived from tropoelastin, ELPs are biocompatible, non-toxic, and non-immunogenic. The unique properties of ELPs have made them a desirable class of fusion tags used in several biomedical applications including targeted drug delivery and enhancing the half-life of protein drugs. The most significant property of an ELP is that when fused to other proteins, the phase transition property of the ELP is maintained, and the target protein can be purified using the thermally driven property of the ELP. The ELP tag purification process is simple and inexpensive, and involves cycling the protein above and below the transition temperature of the ELP fusion followed by centrifugation to obtain the desired protein, without any need for chromatography. Consequently, using ELPs as a purification tag should be potentially interesting to biopharmaceutical companies who spend a significant percentage of their manufacturing costs on expensive protein purification techniques such as chromatography and filtration. However, ELP tags have not yet been used for commercial protein purification due to some challenges of translating this technique, which has been demonstrated mostly in academic laboratories, to a biotechnology manufacturing environment. The article first reviews the state-of-the-art in protein "ELPylation," and discusses some applications which have benefitted from using ELP as a fusion tag. Then, the article discusses the main advantages of using ELP as a purification tag, and evaluates the remaining hurdles for its implementation in industrial protein production. Biotechnol. Bioeng. 2016;113: 1617-1627. © 2016 Wiley Periodicals, Inc.

Chronic skin wounds are characterized by poor re-epithelialization, angiogenesis and granulation. Previous work has demonstrated that topical stromal cell-derived growth factor-1 (SDF1) promotes neovascularization, resulting in faster re-epithelialization of skin wounds in diabetic mice. However, the clinical usefulness of such bioactive peptides is limited because they are rapidly degraded in the wound environment due to high levels of proteases. Here, we describe the development of a recombinant fusion protein comprised of SDF1 and an elastin-like peptide that confers the ability to self-assemble into nanoparticles. The fusion protein and recombinant human SDF1 showed similar binding characteristics, as indicated by the measured equilibrium dissociation constant (Kd) for the binding of free SDF1 or the fusion protein to the CXCR4 receptor. The biological activity of SDF1-ELP, as measured by intracellular calcium release in HL60 cells was dose dependent, and also very similar to that of free SDF1. In contrast, the biological activity of SDF1-ELP in vivo was significantly superior to that of free SDF1. When applied to full thickness skin wounds in diabetic mice, wounds treated with SDF1-ELP nanoparticles were 95% closed by day 21, and fully closed by day 28, while wounds treated with free SDF1, ELP alone, or vehicle were only 80% closed by day 21, and took 42 days to fully close. In addition, the SDF1-ELP nanoparticles significantly increased the epidermal and dermal layer of the healed wound, as compared to the other groups. These results indicate SDF1-ELP fusion protein nanoparticles are promising agents for the treatment of chronic skin wounds.

Objective:In previous work, we demonstrated the development of a novel fusion protein containing stromal cell-derived growth factor-1 alpha juxtaposed to an elastin-like peptide (SDF1-ELP), which has similar bioactivity, but is more stable in elastase than SDF1. Herein, we compare the ability of a single topical application of SDF1-ELP to that of SDF1 in healing 1 · 1 cm excisional wounds in diabetic mice. Approach: Human Leukemia-60 cells were used to demonstrate the chemotactic potential of SDF1-ELP versus SDF1 in vitro. Human umbilical vascular endothelial cells were used to demonstrate the angiogenic potential of SDF1-ELP versus SDF1 in vitro. The bioactivity of SDF1-ELP versus SDF1 after incubation in ex-vivo diabetic wound fluid was compared. The in-vivo effectiveness of SDF1-ELP versus SDF1 was compared in diabetic mice wound model by monitoring for the number of CD31+ cells in harvested wound tissues. Results: SDF1-ELP promotes the migration of cells and induces vascularization similar to SDF1 in vitro. SDF1-ELP is more stable in wound fluids compared to SDF1. In vivo, SDF1-ELP induced a higher number of vascular endothelial cells (CD31+ cells) compared to SDF1 and other controls, suggesting increased vascularization. Innovation: While growth factors have been shown to improve wound healing, this strategy is largely ineffective in chronic wounds. In this work, we show that SDF1-ELP is a promising agent for the treatment of chronic skin wounds. Conclusion: The superior in vivo performance and stability of SDF1-ELP makes it a promising agent for the treatment of chronic skin wounds.

Stromal cell-derived growth factor-1 (SDF1) is a chemokine that is over-expressed at sites of injury and is believed to play an important role in wound repair. At the cellular level, SDF1 regulates the mobilization and trafficking of endothelial progenitors that originate in the bone marrow and functionally contribute to neovascularization and angiogenesis in the wound. Consequently, SDF1 is a potentially interesting therapeutic with the potential to enhance these processes in acute and chronic injuries that otherwise tend to heal poorly, such as spinal cord injury, stroke, myocardial infarction, diabetic skin wounds and acute burns. However, the therapeutic usefulness of SDF1, as many other similar peptide-based growth factors and chemokines, is severely limited due to its short in vivo half-life, as it is rapidly degraded by proteases, which are typically very abundant at the wound site. Several studies have reported methodologies to increase SDF1 in vivo stability by mutating the protease cleavage sites of the molecule. Another approach has been to incorporate the chemokine into biomaterials that shield it from degradation. Yet another approach would be to develop a system that is inherently stable and could be combined with these aforementioned strategies. For example, self-assembled nanoparticles could shield SDF1 (or one of its forms engineered to be more resistant to proteolysis) from proteolysis and then be incorporated into suitable biomaterials. Nanotechnology-based delivery systems have however been used to a very limited extent for SDF1. This paper aims to provide a summary of the various stabilization and delivery methods available for SDF1, some of which have been already used, as well as others that have been used with other bioactive peptides, but would be potentially applicable to SDF1.

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