Directed Evolution of Brain-Derived Neurotrophic Factor for Improved Folding and Expression in Saccharomyces cerevisiae

Abstract

cBrain-derived neurotrophic factor (BDNF) plays an important role in nervous system function and has therapeutic potential. Microbial production of BDNF has resulted in a low-fidelity protein product, often in the form of large, insoluble aggregates incapable of binding to cognate TrkB or p75 receptors. In this study, employing Saccharomyces cerevisiae display and secretion systems, it was found that BDNF was poorly expressed and partially inactive on the yeast surface and that BDNF was secreted at low levels in the form of disulfide-bonded aggregates. Thus, for the purpose of increasing the compatibility of yeast as an expression host for BDNF, directed-evolution approaches were employed to improve BDNF folding and expression levels. Yeast surface display was combined with two rounds of directed evolution employing random mutagenesis and shuffling to identify BDNF mutants that had 5-fold improvements in expression, 4-fold increases in specific TrkB binding activity, and restored p75 binding activity, both as displayed proteins and as secreted proteins. Secreted BDNF mutants were found largely in the form of soluble homodimers that could stimulate TrkB phosphorylation in transfected PC12 cells. Site-directed mutagenesis studies indicated that a particularly important mutational class involved the introduction of cysteines proximal to the native cysteines that participate in the BDNF cysteine knot architecture. Taken together, these findings show that yeast is now a viable alternative for both the production and the engineering of BDNF.

Nanoformulation of Brain‐Derived Neurotrophic Factor with Target Receptor‐Triggered‐Release in the Central Nervous System

“…Consistent with prior reports, [9] native BDNF forms large and highly heterogeneous aggregates with an effective diameter ( D eff ) of about 600 nm and a polydispersity index (PDI) greater than 0.4 (Figure 1a,b). Therefore, it cannot be used as an injectable pharmaceutical agent without proper formulation.…”

Pro region engineering of nerve growth factor by deep mutational scanning enables a yeast platform for conformational epitope mapping of anti‐NGF monoclonal antibodies

“…A major limitation of the method is the requirement that the displayed target protein be in a conformation recognizable by the antibody. This is an issue with cNGF and consistent with a previous study showing that the related neurotrophin BDNF displayed on the surface of yeast in a mostly inactive conformation (Burns et al, ).…”

Determination of binding affinity upon mutation for type I dockerin–cohesin complexes from C lostridium thermocellum and C lostridium cellulolyticum using deep sequencing

“…While assumptions 1–4 can be externally validated or are otherwise reasonable for many protein–protein interactions, the latter two assumptions are known to be simplifications. For example, in certain cases, protein variants have been shown to have very different average surface display . Additionally, intrinsic counting error (also known as Poisson noise) decreases with increasing sequencing depth.…”

“…For example, in certain cases, protein variants have been shown to have very different average surface display. 45,46 Additionally, intrinsic counting error (also known as Poisson noise) decreases with increasing sequencing depth. Accordingly, one would expect that the error in determining the frequency of a variant sequenced 10 times in the reference population would be greater than that of a variant represented 1000 times.…”