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Expression and Purification of Neurotrophin-Elastin-Like Peptide Fusion Proteins for Neural Regeneration

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Abstract

Background

Neural injuries such as spinal cord injuries, traumatic brain injuries, or nerve transection injuries pose a major health problem. Neurotrophins such as nerve growth factor (NGF) or brain-derived neurotrophic factor (BDNF) have been shown to improve the outcome of neural injuries in several pre-clinical models, but their use in clinics is limited by the lack of a robust delivery system that enhances their bioavailability and half-life.

Objectives

We describe two fusion proteins comprising NGF or BDNF fused with elastin-like peptides (ELPs). The aim of this study was to investigate the biological activity of neurotrophin-ELP (N-ELP) fusion proteins via in vitro culture models.

Methods

NGF and BDNF were cloned in front of an elastin-like polypeptide sequence V40C2. These proteins were expressed in bacteria as inclusion bodies. These fusion proteins underwent solubilization via 8 M urea and purification via inverse transition cycling (ITC). We measured the particle size and the effect of temperature on precipitated particles using dynamic light scattering (DLS). We used western blot analysis to confirm the specificity of NGF-ELP to tropomyosin receptor kinase A (TrkA) antibody and to confirm the specificity of BDNF-ELP to TrkB antibody. PC12 cells were used to perform a neurite outgrowth assay to determine the biological activity of NGF-ELP. Bioactivity of BDNF-ELP was ascertained via transfecting human epithelial kidney (HEK 293-T) cells to express the TrkB receptor.

Results

The proteins were successfully purified to high homogeneity by exploiting the phase transition property of ELPs and urea, which solubilize inclusion bodies. Using PC12 neurite outgrowth assay, we further demonstrated that the biological activity of NGF was retained in the fusion. Similarly, BDNF-ELP phosphorylated the TrkB receptor, suggesting the biological activity of BDNF was also retained in the fusion. We further show that owing to the phase transition property of ELPs in the fusion, these proteins self-assembled into nanoparticles at their respective transition temperatures.

Conclusion

These fusion proteins are useful for neural regeneration, as they not only retain the biological activity of the neurotrophin but also self-assemble into nanoparticles, thereby simultaneously serving as drug-delivery vehicles. These nanoparticles can serve as drug depots and will increase bioavailability by limiting neurotrophin loss due to diffusion, thereby allowing controlled spatio-temporal delivery of the neurotrophin.

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Acknowledgments

The authors acknowledge Yuan Yuan, Raul Iglesias, and Bernard Batson.

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Authors and Affiliations

  1. Department of Chemical and Biomedical Engineering, University of South Florida, 4202 E Fowler Avenue, ENB 118, Tampa, FL, 33620, USA

    Tamina Johnson & Piyush Koria

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  1. Tamina Johnson
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  2. Piyush Koria
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Corresponding author

Correspondence to Piyush Koria.

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Conflicts of interest

Tamina Johnson and Piyush Koria have no conflicts of interest that are directly relevant to the content of this study.

Funding

This work was funded by startup funds from the University of South Florida (PK) and the new researcher grant (PK) from University of South Florida, and in part by Institutional Research Grant number 93-032-16 from the American Cancer Society (PK). Tamina Johnson was supported by NSF Florida Georgia Louis Stokes Alliance for Minority Participation Bridge to the Doctorate Award HRD #1139850, the USF Graduate Student Success Diversity Fellowship, and The Alfred P. Sloan Minority Ph.D. Program.

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Johnson, T., Koria, P. Expression and Purification of Neurotrophin-Elastin-Like Peptide Fusion Proteins for Neural Regeneration. BioDrugs 30, 117–127 (2016). https://doi.org/10.1007/s40259-016-0159-4

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