Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family and is involved in neuronal cell survival, differentiation, growth cessation, and apoptosis. Because of these attributes, BDNF has the potential to be a viable therapeutic for neurological diseases such as stroke, Alzheimer's disease and Parkinson's disease. However, the difficulties and costs associated with its production limit the feasibility of using BDNF or other neurotrophins as therapeutics. To this end, we have employed yeast surface display combined with directed evolution approaches to improve BDNF display and production using yeast. In its native form, surface-displayed and secreted BDNF product in all cases was found largely in the form of a disulfide-bonded aggregate, and was in some way capable of evading the yeast quality control machinery. Therefore, the BDNF protein itself was engineered in an attempt to create a protein product that folded with higher fidelity. Multiple rounds of random mutagenesis and gene shuffling were used to create surface-displayed BDNF that is active and avidly binds its natural ligands, p75 and TrkB. Mutations could be classified based on structural locale and indicated that the cysteine knot architecture characteristic to neurotrophins plays an important role. When secreted as a fusion to the cell surface display tether, Aga2p, the mutant BDNF constructs no longer form disulfide-bonded aggregates and are expressed as active proteins at levels well in excess of that found with wild-type BDNF. Thus, the engineered BDNF-Aga2p fusion allows proper folding and disulfide bond formation in BDNF and provides a novel platform for neurotrophin engineering.

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