Directed evolution of brain-derived neurotrophic factor for improved folding and expression in Saccharomyces cerevisiae

Abstract

Brain-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.

FIG 1

Production of BDNF using yeast. (A) Schematics of BDNF constructs displayed on the yeast surface as a dimer of two surface display proteins (i) and as a dimer of one surface display protein and one secreted and captured protein (ii). (B) Expression and receptor-binding properties of wild-type BDNF displayed on the yeast surface using scheme i. Shown are sample flow cytometric dot plots of surface-displayed wild-type BDNF either colabeled with its natural receptor TrkB and an anti-c-myc antibody to monitor full-length expression or labeled with p75 alone. The negative sample is yeast displaying an irrelevant single-chain antibody labeled in the exact same manner as the BDNF sample. Note the presence of a full-length single-chain antibody on the yeast surface but the absence of TrkB and p75 labeling. Quantified data are reported in Table 2. (C) Western blots of secreted wild-type BDNF with (+) or without (−) reduction by DTT. Supernatants from triplicate independent transformants were analyzed. Molecular sizes (in kDa) are indicated on the left.

FIG 2

Directed evolution of BDNF. (A) Flow chart of the directed-evolution process showing outcomes and screening criteria. (B) Flow cytometric data illustrating the directed-evolution improvement in BDNF activity (TrkB and p75) and full-length expression (anti-c-myc antibody) as a surface-displayed protein. Mutant T5 is shown as an example of DE Rd1 mutants and mutant K8 as an example of DE Rd2 mutants. Quantified data for all DE Rd1 and Rd2 mutants assessed are displayed in Table 2.

FIG 3

Comparison of the binding and expression properties of displayed and secreted BDNF proteins. (A) Specific binding activity toward the TrkB receptor as measured on the yeast surface by flow cytometry (Display) or for the secreted protein by ELISA (Secretion). TrkB binding activity is expressed per full-length molecule as assessed by the c-myc epitope and normalized to wild-type BDNF activity. (B) Specific binding activity toward the p75 receptor, determined as described for the TrkB receptor, and normalized to the activity of the T5 BDNF mutant. (C) Relative expression levels determined by flow cytometry (Display) or Western blotting (Secretion) and normalized to wild-type BDNF expression. In panels A to C, data are expressed as means ± standard deviations for triplicate independent transformants. Statistical evaluation was performed using an unpaired, two-tailed Student t test. (D) Sample Western blot data used to generate the relative secretion values reported in panel C.

FIG 4

Structural locations of identified mutations. (A) BDNF (yellow) and neurotrophin 4 (blue) heterodimer structure (PDB code 1HCF [42]). Residues subject to mutation in P10 and K8 are highlighted. (B) Enlarged view of cysteine knot protein core highlighting the native Cys13 and Cys68 residues as well as the Ser11Cys and Gly67Cys mutations, along with the three intramolecular disulfide bonds (red lines). (C) Structure of the neurotrophin 4 homodimer (dark and light green) binding to the TrkB receptor (blue) (PDB code 1B8M [10]). The homologous NT4 residues corresponding to the P10 and K8 mutations are highlighted (red and orange).

FIG 5

Evaluation of cysteine mutations by site-directed mutagenesis and yeast surface display. Cysteine mutations and reversions were incorporated into the P3, K8, and wild-type BDNF (WT) constructs. Specific binding activities toward p75 and TrkB, along with full-length expression levels (c-myc), were measured by flow cytometry. Data are means ± standard deviations for triplicate independent transformants. Statistical evaluation was performed using an unpaired, two-tailed Student t test. *, labeling not detected for this construct.

FIG 6

Surface capture of secreted BDNF mutants. Surface display and secretion constructs were coexpressed as indicated in Fig. 1Aii prior to evaluation of TrkB binding and of c-myc (tethered display) and His₆ (secreted and captured display) epitope tags by flow cytometry. All labeling is normalized to that for the K8 mutant. The tethered c-myc sample is an Aga2p fusion with the c-myc epitope (i.e., Fig. 1Aii without the BDNF insertion). The secretion sample (4-4-20) represents an anti-fluorescein single-chain antibody. *, His₆ labeling not detected for these constructs. Data are means ± standard deviations for triplicate independent transformants.

FIG 7

Size exclusion chromatography analysis of the secreted and purified K8 BDNF mutant. (A) (Top) Elution chromatogram of molecular size standards. (Bottom) Western blot of K8 sample eluates aligned by elution time with the molecular size standards. Samples were run under reducing conditions with (+) or without (−) glutaraldehyde (G.A.) cross-linking. (B) Western blot of secreted and purified K8 under both reducing and nonreducing SDS-PAGE conditions (with and without DTT) and with and without cross-linking (with and without G.A.). A slightly overexposed blot is presented to allow viewing of all BDNF species.

FIG 8

TrkB phosphorylation by the K8 and P10 BDNF mutants. TrkB-transfected PC12 cells were exposed to commercial wild-type BDNF (Std), the BDNF mutant K8, the BDNF mutant P10, and two negative controls: a yeast supernatant from cells containing an empty secretion vector (designated 316) and a saline-treated sample (PBS). Western blotting of the pan-Trk-immunoprecipitated product with detection of either the phosphorylated TrkB receptor (P-TrkB) or the total Trk receptor (Trk) was performed. Bands corresponding to the full-length TrkB receptor at ∼145 kDa are shown.