recombinant full length human tau  (MedChemExpress)

Journal: bioRxiv

Article Title: 14-3-3 binding regulates Tau assembly and microtubule association

doi: 10.1101/2024.03.15.585148

Figure Lengend Snippet: a, Domain structure of the longest human Tau isoform (2N4R, 441 aa) consisting of the N-terminal projection domain with two N-terminal inserts (N1, N2), the proline-rich domain (P1+P2), and the C-terminal microtubule binding region that includes four pseudo-repeats (R1-R4) and short sequences up- and downstream of these. Phosphorylation of serine residues S214 and S324 enables 14-3-3ζ binding to Tau. Domain structure of phospho-peptide Tau-pS214/pS324 (pS2) is shown as well (38aa; Tau 210-222(pS214) -GGGSGGGSGGG-Tau 318-331(pS324) . b, Fluorescent anisotropy measurements for the binding of Tau-pS214/pS324, Tau-pS14, and Tau-pS324 peptides to 14-3-3ζ. 14-3-3ζ was titrated into 10 nM of respective fluorescein-labeled Tau peptide. Data shown as mean±SD, representative experiment with N=3 technical replicates (see for two more experiments). c, Cartoon showing semi-transparent surface of the 14-3-3ζ dimer (gray) complexed with the pS214 and pS324 binding sites of the tau peptide (green rods). d, Top view of pS214 and pS324 binding sites (green rods) into the 14-3-3ζ binding grooves (grey semi-transparent surface). Green dash line shows connective amino acid residues between the binding sites. e+f, Close-up of the binding grooves of monomer A and B (grey surface) containing the tau peptide binding sites (green rods). Final 2Fo-Fc electron density map of the peptide is shown (blue mesh, contoured at 1s). g, Surface charge distribution on 14-3-3 ζ dimers mapped on the crystal structure shown in e and f. Negatively charged areas are indicated in red, and positively charged ones in blue. Note that the binding pocket of the Tau phospho-peptide, pS2, is positively charged, whereas much of the outer surface of 14-3-3ζ is negatively charged.

Article Snippet: Recombinant human full-length Tau (2N4R, hTau40) and Tau ΔK280 were expressed in E. coli BL21 Star (DE3) (Invitrogen) cells as previously described and purified following an established protocol .

Techniques: Binding Assay, Labeling

Journal: bioRxiv

Article Title: 14-3-3 binding regulates Tau assembly and microtubule association

doi: 10.1101/2024.03.15.585148

Figure Lengend Snippet: a, Schematic: Full-length Tau binding to 14-3-3ζ depends on phosphorylation at serine residues S214 and S324. b, Western blots of recombinant Tau variants in vitro phosphorylated using PKA kinase (Tau variants: wildtype Tau, Tau S214A, Tau S324A, Tau S214A-S324A (Tau S2A)) using antibodies specific for phosphorylated residues in Tau (Tau pS214 and Tau pS324) and total Tau. c, Thermal shift assay of PKA-phosphorylated and non-phosphorylated Tau variants binding to 14-3-3ζ. PKA-phosphorylation significantly increases binding of Tau to 14-3-3ζ, which is reduced upon S>A mutation of the relevant phosphorylation sites, S214A and S324A. Mutation of both serine residues abolishes Tau binding. Data shown as mean±SD, N=3 independent experiments. One-way ANOVA with Tukey post-test.

Article Snippet: Recombinant human full-length Tau (2N4R, hTau40) and Tau ΔK280 were expressed in E. coli BL21 Star (DE3) (Invitrogen) cells as previously described and purified following an established protocol .

Techniques: Binding Assay, Western Blot, Recombinant, In Vitro, Thermal Shift Assay, Mutagenesis