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 cellular and protein engineering approaches to improve BDNF production in yeast. Since BDNF is a cysteine-knot protein possessing three intramolecular disulfide bonds, yeast proteins involved in the disulfide bond formation pathway, Ero1p and PDI, were overexpressed in an effort to enhance secretion. In addition, as BDNF is a natural homodimer formed via a highly hydrophobic interface, the chaperone, BiP was also overexpressed. Only Ero1p promoted any increase in BDNF expression (1.6-fold), while BiP had little effect, and PDI hindered expression. Interestingly, the 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. In this way, using random mutagenesis and directed evolution, BDNF was engineered for active production using yeast surface display approaches. Multiple rounds of mutagenesis and gene shuffling were used to create surface-displayed BDNF that is active and avidly binds its natural ligands, p75 and TrkB. 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 at levels in 2- to 5-fold 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.