Pmal c2x vector

DNA sequences coding for Pgm, Ku70a and Ku80c were obtained by gene synthesis (DNA 2.0 or Eurofins MWG/Operon). The synthetic PGM DNA sequence was first cloned into the pMAL-c2x vector (New England Biolabs). The MBP-PGM fusion, the MBP, the KU70a and the KU80c sequences were further introduced into the pVL-1392 vector (BD Biosciences). A HA tag was added to the C-terminus of Ku70a and the N-terminus of Ku80c. All plasmid sequences are provided in a supplementary information file (Text S1). Plasmids pVL1392-MBP-PGM, pVL1392-MBP, pVL1392-KU70a-HA and pVL1392-HA-KU80c were transfected individually into High Five cells together with the BD BaculoGold Linearized Baculovirus DNA (BD Biosciences).

The genes encoding the variable heavy (vH) and variable light (vL) chain were generated by reverse transcription-polymerase chain reaction from total RNA of the B2.1A-secreting hybridomas cells. The vH and vL segments were linked via a nucleotide sequence encoding a flexible linker (GGSGGSGGGGSGGGGSGGGAS) and this cassette placed downstream from a Igk-leader sequence in a modified mammalian expression vector pDisplay (Life Technologies) to generate a single-chain variable fragment (scFv) expression construct. The vH-linker-vL cassette was subsequently sub-cloned into pLOU3, an Escherichia coli expression vector derived from the pMAL-c2x vector (NEB). The construct encodes a maltose-binding protein (MBP)-B2.1 A scFv fusion protein, which carries a 6-histidine sequence at its N-terminus and a tobacco etch virus (TEV) protease cleavage site at the C-terminus of MBP. Briefly, the fusion protein was expressed in E. coli strain Rosetta-gami 2(DE3)pLysS (EMD Millipore) and the protein purified from cell extract using HisTrap and MBPTrap columns (GE Healthcare). The protein was then digested with TEV protease and re-applied to the HisTrap column. The unbound fraction containing B2.1 A scFv was further purified on a 16/60 Sephacryl S100 (GE Healthcare) in PBS.

For cell-free protein degradation assays, full-length MOC1 was amplified using primers (Supplementary Table 2) and cloned into the pMAL-c2x vector (NEW ENGLAND BioLabs, Catalog no. E8000S). Recombinant MBP-MOC1 proteins were expressed in E. coli BL21 (DE3) cells and purified through Amylose Resin (NEW ENGLAND BioLabs, Catalog no. E8021S). Seedlings of GFPOE, SLR1-GFPOE, sd1, GID1OE, and the corresponding WT were grown in a SANYO MLR Plant Growth Chamber (MLR-351) for 14 days at 28 °C during daytime and 25 °C at night with a 16:8 (day:night) photoperiod. The shoot bases (0–0.5 cm aboveground) were collected, and total proteins were extracted with degradation buffer (25 mM Tris-HCl, pH 7.5, 10 mM NaCl, 10 mM MgCl₂, 4 mM PMSF, 5 mM dithiothreitol, and 10 mM ATP)^{42 (link)} and adjusted to equal concentrations with the degradation buffer. Then 100 ng of purified MBP-MOC1 or His-trx-SLR1 protein was incubated in 100-μL extracts (containing 500 μg of total proteins) for the individual assays. The extracts were incubated at 28 °C, and samples were taken at the indicated intervals for protein blotting. Anti-MBP (NEW ENGLAND BioLabs, Catalog no. E8032), anti-His (Abmart, Catalog no. M20020), and anti-RPN6 (Enzo Life Sciences, Catalog no. BML-PW8370) antibodies were used at a 1:20,000 dilution.