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. 2023 Jun 22;299(8):104948. doi: 10.1016/j.jbc.2023.104948

Figure 5.

Figure 5

hRpn1-biotin is an effective handle for purification of 26S proteasomes.A, schematic representation of the workflow for purification of 26S proteasomes (RP in orange, CP in gray) from C1 cells by hRpn1-biotin (red star). CRISPR/Cas9-engineered C1 cells were seeded in culture dishes (step 1) and the WCE post lysis (step 2) incubated with neutravidin resin for 1 h at 4 °C (step 3). After four washing steps, the mixture was treated with His-tagged TEV protease (step 4) for 1 h at 21 °C to release 26S proteasomes from the resin by cleaving hRpn1 from biotin. The eluant was incubated with TALON resin to remove His-tagged TEV protease (step 5) and the resin sedimented by centrifugation. Supernatant containing purified 26S proteasomes was flash frozen in liquid nitrogen and stored at −80 °C. B, immunoblot to evaluate the presence of 26S proteasomes by using hRpn1, hRpt5, and β5 antibodies before and after treatment with TEV protease. WCE from parental WT and C1 cells is also included. C, purified 26S proteasomes from the protocol described in (A) were evaluated by SDS-PAGE (4–12%) with SYPRO Ruby Fluorescent stain. Commercial proteasomes were included for comparison. D, purified and commercial 26S proteasomes were subjected to native-PAGE and evaluated with SYPRO Ruby Fluorescent stain. E, representative image of raw electron micrograph of negatively stained 26S proteasomes purified from C1 cells captured at 36k magnification, with a scale bar corresponding to 50 nm. The insert shows an expanded region captured at 92k magnification, with a 20 nm scale bar. Different orientations of 26S and CP proteasomes are highlighted with purple-colored boxes and circles, respectively. F, C1-purified proteasomes are active against ubiquitinated p53. Ubiquitinated p53 was incubated with C1 or commercial proteasomes for the indicated time points, after which the mixture was separated by SDS-PAGE and immunoblotted with anti-p53 or anti-β5 antibodies (left). Quantification of p53 normalized to β5 at the indicated time points following incubation with C1-purified (black squares) or commercial (gray circles) proteasomes plotting the mean value (n = 3) from three independent experiments with standard deviation (right). CP, core particle; RP, regulatory particle; TEV, tobacco etch virus; WCE, whole cell extract.