Superdex s200 16 60

Full-length WT hTRAP1 and zTRAP1 (without mitochondrial signal sequence) was cloned into a pET151DTOPO vector as described previously (19 (link)). Point mutations were introduced via site-directed mutagenesis by PCR. For FRET assays, single cysteines (G151C and K428C) were introduced into a cysteine-free zTRAP1 construct (21 (link)) to enable site-specific labeling with maleimide derivatives of Alexa Fluor 555/647 (Life Technologies). Proteins were expressed in BL21(DE3)-RIL Escherichia coli cells grown in TB media by addition of 0.5 mm isopropyl 1-thio-β-d-galactopyranoside at A₆₀₀ ∼0.8, then incubated with shaking for 12–18 h overnight at 16 °C. All of the TRAP1 constructs have an N-terminal His₆ tag with a cleavable tobacco etch virus site. Purification follows a standard protocol for nickel-nitrilotriacetic acid chromatography using 40 mm Tris buffer at pH 8.0, followed by an anion exchange with a MonoQ column, and a final size-exclusion step with a Superdex S200 16/60 (GE Healthcare) column.

Monomeric fractions of N were pooled and complexed with purified VHH (ratio N:VHH = 1:1.5). After 1 h incubation at 4°C, size-exclusion chromatography (SEC) was applied to separate the N–VHH complex from free VHH using Superdex S200 16/60 (GE Healthcare) equilibrated with 10 mM Tris pH 8.0, 150 mM NaCl. The purified complex (N and VHH) was concentrated to 13 mg ml⁻¹ and stored at 4°C.

Characterization of Δ133p53β was done by SEC and DLS. SEC was performed on a Superdex S200 16/60 (HiLoad 16/600 Superdex 200 pg, GE Lifescience) on an AKTA Pure system (GE Lifescience). The protein was concentrated and injected on the column with a 5 ml loop equilibrated with buffer A. DLS was performed using the Zetasizer NaNo-S system (Malvern Instruments) on the SEC maximum peak. Data were collected in 3 consecutive runs of 15 repeated measurements at 20 °C. The sample volume used was 150 µl at 0.8 mg/ml.