Pdest17 expression vector

A complete AtADPGP encoding cDNA from the Arabidopsis Biological Center at Ohio State University [73] (link) was cloned into the pDEST17 expression vector (Invitrogen) to create pDEST17-AtADPGP (Figure S2). BL21 C43 (DE3) cells transformed with pDEST-AtADPGP were grown in 100 ml of liquid LB medium to an absorbance at 600 nm of 0.5 and then 1 mM IPTG was added. After 5 h, cells were centrifuged at 6,000 g for 10 min. The pelleted bacteria were resuspended in 6 ml of His-bind binding buffer (Novagen), sonicated and centrifuged at 10,000 g for 10 min. The supernatant thus obtained was subjected to His-bind chromatography (Novagen). The eluted His-tagged AtADPGP was then rapidly desalted by ultrafiltration on Centricon YM-10 (Amicon, Bedford, MA).
The purified recombinant AtADPGP was electrophoretically separated by 12% SDS-PAGE and stained with Coomassie Blue. A ca. 38 kDa protein band was eluted and utilized to produce polyclonal antisera by immunizing rabbits.
For immunoblot analyses, samples were separated on 10% SDS-PAGE, transferred to nitrocellulose filters, and immunodecorated by using the antisera raised against either AtADPGP or EcASPP [27] (link) as primary antibody, and a goat anti-rabbit IgG alkaline phosphatase conjugate (Sigma) as secondary antibody.

The coding sequence regions for P53 (NM_001276760.1), NY-ESO-1 (NM_001327.2), PRDX6 (NM_004905.2), BMI1 (NM_005180.8), MMP7 (NM_002423.3), and HSP70 (NM_005345.5) were subcloned into the pDEST17 expression vector (Invitrogen, Waltham, MA). We conducted the expression, purification, and analysis of these recombinant proteins as described in our previous studies [13 (link), 14 (link)].

PtUGEc was introduced into pDEST17 expression vector (Invitrogen) containing an N-terminal 6xHis-tag and an IPTG inducible promoter. Gene expression in BL21 Star cells (Invitrogen) was induced by adding IPTG to a final concentration of 1 mM, and cultures were grown at 18°C overnight. PtUGEc was purified from the supernatant of lysed cell pellets using HIS-Select Nickel Affinity Gel purification (Sigma-Aldrich). Lysis of bacterial cells took place using CelLytic B 2X containing 0.2 mg/ml lysozyme, 50 U/ml benzonase (all Sigma-Aldrich) and proteinase inhibitor cocktail (Roche). HIS-tagged PtUGEc was desalted with PD-10 desalting columns (Amersham Biosciences). Samples of 30 μg His-PtUGEc protein were separated on a Novex 8-16% Tris Glycine Gradient gel (Invitrogen) and stained with Coomassie Brilliant Blue.