E coli bl21 de3 plyss cells

Escherichia coli DH5α cells (Agilent, USA) were used for initial cloning, while E. coli BL21 (DE3)pLysS cells (Agilent, Santa Clara, CA, USA) were utilised for the heterologous expression of the recombinant M. pachydermatisβ-CA. The pET100/D-TOPO vector was purchased from Invitrogen (Carlsbad, CA) with the feature to express the recombinant protein as a fusion protein with a 6-histidine tag at the N-terminus. Luria Bertani Broth (LB), ampicillin, and other chemicals were obtained from Merck (Darmstadt, Germany).

GST or GST-ATG8 proteins were expressed (from GST-ATG8 (pAL) plasmids) in E. coli BL21 (DE3) plysS cells (Agilent) in LB medium supplemented with 50 µg/ml kanamycin. Expression was induced by addition of 0.5 mM IPTG at OD₆₀₀ = 0.6 and cells were incubated at 25 °C overnight or at 37 °C for 5 h. Harvested cells were lysed in 50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 0.1% Triton X-100, 0.4 mM AEBSF and 15 µg/ml Benzamidine. Fusion protein was batch-adsorbed onto Glutathione-Sepharose 4B beads (GE Healthcare). After five washes with wash buffer (50 mM Tris, pH 8.0, 250 mM NaCl, 0.4 mM AEBSF and 15 µg/ml benzamidine) fusion proteins were eluted in 50 mM Tris pH 8.0, 2 mM L-glutathione reduced, 0.4 mM AEBSF and 15 µg/ml benzamidine.
A MultiPep or ResPep SL automated synthesizer (INTAVIS Bioanalytical Instruments AG, Germany) was used for SPOT synthesis of peptide arrays on cellulose membranes.⁷⁰ (link) After blocking membranes in TBST with 5% nonfat dry milk, peptide interactions with GST or GST fusion proteins were tested by overlaying the membranes with either 1 µg/ml (mutational peptide array scan) or 2 µg/ml of recombinant protein (all other peptide arrays) for 2 h at room temperature. Membranes were washed in TBST, and bound proteins were detected with HRP-conjugated anti-GST antibody (1:5000, GE Healthcare, RPN1236).

Vectors encoding WT, x2, x5, and x11 Flucs in pET16b were prepared in previous work. 10X-N-terminal His-tagged Eluc and CBR were amplified from pLR6-Eluc (GenBank KU756582.1), provided kindly by Mikhail Koksharov (Brown University, United States), or pGex-CBR plasmids, respectively, and cloned into the pET16b with NcoI and BamHI. These were transformed into E. coli BL21 (DE3) pLysS cells (Agilent Technologies, CA, United States) and over-expressed and purified by nickel-NTA affinity chromatography as described previously (Law et al., 2006 (link); Jathoul et al., 2012a ). The pCCL vector 305 was kindly provided by Prof. Riccardo Brambilla (Cardiff University, Cardiff, United Kingdom) under MTA.