Full length s protein constructs used for expression

Full length S protein constructs used for expression in mammalian cells: a
sequence encoding SARS-CoV-2 S was codon optimised for expression in
mammalian cells and cloned into pcDNA3.1 + (modi fi ed to be compatible with the
PiggyBac transposase system) using the restriction sites NheI and NotI. Where
indicated, an HA tag was inserted after the signal peptide by introduction into the
forward primer, ampli fi cation by PCR and insertion into pcDNA3.1 + . Key
residues in the cytoplasmic tail of S were mutated as indicated by introducing
mutations into primers, ampli fi cation of a small region at the 3 ′ end of the gene
and insertion using the restriction sites BstEII and NotI.
Mammalian cell culture . Human embryonic kidney 293T (ATCC, CRL-3216),
U2OS (ATCC, HTB-96) and Vero (ATCC, CCL-81) cells were cultured in Dul-
becco ’ s modi fi ed Eagle ’ s medium Glutamax (DMEM; Gibco) supplemented with
10% fetal calf serum (FCS) and penicillin/streptomycin at 37 °C and 5% CO
2
.
Unless indicated otherwise, cells were transfected using polyethylenimine (PEI;
Polyscience, 24765) dissolved in PBS to 1 mg/ml. The ratio of PEI (μL) to DNA
(μg) used was 3:1; PEI was dissolved in Opti-Mem, incubated at room temperature
for 5 min, DNA was added and incubated for another 15 – 20 min at room tem-
perature before dropwise addition onto cells which had been seeded the day before.
Cells were not authenticated after being obtained from the ATCC, but were free of
mycoplasma as determined by regular testing (MycoAlert, Lonza).
Protein expression in bacteria . GST (pGEX6p2 vector), the different versions of
GST-S constructs and the His6-tagged interactors were expressed as follows:
plasmids were transformed into E. coli BL21-CodonPlus (DE3)-RIL (Agilent,
230245). From an overnight starter culture, cells were grown in 2xTY medium
containing 100 μg/mL ampicillin (or 50 μg/mL Kanamycin for His6-VPS26) and
34 μg/mL chloramphenicol at 37 °C in a shaking incubator. When the culture
reached OD
600
= 0.6 - 0.8, the temperature was lowered to 16 °C, protein
expression was induced with 100 μM of Isopropyl β -D-1-thiogalactopyranoside
(IPTG), and incubated overnight. Bacteria cells were harvested by centrifugation at
4000 × g at 4 °C for 15 min and were mechanically resuspended on ice in lysis
buffer containing 50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 5 mM 2-
mercaptoethanol, 1% Triton X-100, and supplemented with protease inhibitor
cocktail (cOmplete, Roche). Cells were lysed by sonication and the lysates were
clari fi ed by centrifugation at 20,000 × g at 4 °C for 15 min. Clari fi ed lysates were
fl ash frozen in liquid nitrogen and thawed as needed for the binding assays.
GST-pulldowns using 293T cell lysates . Pull downs for mass spectrometry :
clari fi ed lysates from 450 mL 2xTY cultures containing bacteria expressing
recombinant GST, GST-S tails (product of pJC149, pJC150, pJC247) were thawed.