title: Expression and Purification of Neurotrophin-Elastin-Like Peptide Fusion Proteins for Neural Regeneration.
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.
  
  
  OBJECTIVE
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Chemical and Biomedical Engineering, University of South Florida, 4202 E Fowler Avenue, ENB 118, Tampa, FL, 33620, USA.
authors.forename: Tamina
authors.initials: T
authors.lastname: Johnson
authors.affiliation: Department of Chemical and Biomedical Engineering, University of South Florida, 4202 E Fowler Avenue, ENB 118, Tampa, FL, 33620, USA. pkoria@usf.edu.
authors.forename: Piyush
authors.initials: P
authors.lastname: Koria
mesh_terms: D000818:Animals; D019208:Brain-Derived Neurotrophic Factor / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D016503:Drug Delivery Systems / Q000379:methods; D004549:Elastin / Q000378:metabolism / Q000494:pharmacology*; D057809:HEK293 Cells; D006801:Humans; D020932:Nerve Growth Factor / Q000378:metabolism; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D016716:PC12 Cells; D010316:Particle Size; D010455:Peptides / Q000378:metabolism / Q000494:pharmacology*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism / Q000494:pharmacology*; D012038:Regeneration / Q000187:drug effects*
chemical_list: D019208:Brain-Derived Neurotrophic Factor; D010455:Peptides; D011993:Recombinant Fusion Proteins; C579455:BDNF protein, human; D004549:Elastin; D020932:Nerve Growth Factor