Neurotrophin Engineering for Production In Yeast

Neurotrophins are a family of proteins involved in nearly all neuronal processes including growth, differentiation, and survival.  These proteins draw significant scientific interest because of their potential involvement in neurodegenerative disorders including stroke, epilepsy, Alzheimer’s and Parkinson’s diseases.  While promising compounds, a major factor limiting the development of neurotrophins as therapeutics is their high cost of production.  Neurotrophin production is hindered by a complex protein architecture which is difficult to produce with high fidelity and yield in heterologous systems.  Current neurotrophin production strategies include mammalian and bacterial platforms.  Mammalian cell cultures produce active neurotrophins, but attain relatively low yields.  Bacterial cell cultures produce higher neurotrophin yields but the protein is found in an inactive form which must be isolated and refolded.  To overcome limitations presented by the current mammalian and bacterial neurotrophin production systems, we have employed the yeast, Saccharomyces cerevisiae, for neurotrophin production with brain-derived neurotrophic factor (BDNF) as the model neurotrophin.

A two fold approach was taken.  First the BDNF protein was subjected to directed evolution and screened using yeast surface display technology.  Several mutations that allow proper BDNF folding and processing in yeast were indentified.  The soluble production of these mutants was found to increase 2 to 5-fold in comparison to wild-type, reaching levels of 1 mg/L while still retaining their biological activity (intracellular TrkB phosphorylation).  Second, neurotrophins are naturally produced as pro-protein precursors that are converted into their mature, active forms by proteolytic removal of the pro-peptide.  Interestingly, pro-peptides have been shown to have chaperone-like effects in protein processing by aiding in folding of the active protein structures.  Simply the addition of the native human BDNF pro-peptide improved folding of the BDNF protein in yeast but decreased its production. To improve both folding and production, the pro-peptide was optimized for the yeast secretory environment through protein engineering.  After two rounds of directed evolution, we were able to achieve yeast-specific BDNF pro-peptides which showed 2.5-fold improvement in production and 3-fold improvement in receptor binding over wild-type.  Like the aforementioned BDNF mutants, secreted BDNF products from optimized pro-peptide constructs were biologically active.  Finally, universality of the BDNF and pro-peptide mutations were tested using other members of the neurotrophin family.