E coli bl21 de3

Bacillus clausii DSM8716, Bacillus coagulans DSM1, Marivirga tractuosa DSM4126, Spirosoma linguale DSM74 and Streptomyces pristinaespiralis DSM40338 were obtained from the German collection of microorganisms and cultivated according to the instructions of the supplier (DSMZ, http://www.dsmz.de). E. coli strain JM109 [genotype endA1 recA1, gyrA96, thi, hsdR17,(rK⁻, mK⁺), relA1, supE44, l-, Δ(lac-proAB), (F’, traD36, proAB, lacI^qZΔM15)] was purchased from Promega (Madison, USA). E. coli BL21(DE3) was purchased from Agilent Technologies. Rosetta™ 2(DE3) [genotype F– ompT hsdS_B(rB^– mB^–) gal dcm (DE3) pRARE2 (Cam^R)] was purchased from Merck Millipore (Germany).
E. coli strains were routinely grown in LB medium at 37 °C and 150 rpm. For plasmid selection 100 mg L⁻¹ ampicillin was added to LB agar plates and liquid medium and 34 μg/mL chloramphenicol when cells were co-transformed with pRARE2.

E coli Top 10 with pVR21 plasmid containing norovirus capsid genes were propagated at 30°C in the presence of 0.1mg/ml carbenicillin in Luria-Bertani broth. For full IgG expression and characterization, VH and VL plasmids were transfected into 30 mL cultures of Expi293F cells (Invitrogen) at a 1:2 ratio and incubated at 37°C and 8% CO₂ for 7 days.
For protein crystallization, P-domains were transfected into E.coli BL21 (DE3) (Agilent Technologies) and plated onto Luria-Bertani plates with 50μg/mL Ampicillin (LB-Amp). VH and VL chains were cotransfected into Expi293F cells (ThermoFisher) using a 3:1 ratio of Turbo293 transfection reagent (Speed Biosystem) to plasmid. Cells were incubated at 37°C, 5% CO₂, 130 rpm shaking, 70% humidity for 18 h and subsequently boosted with 80mL of AbBooster (Abi Scientific).

Escherichia coli DH5α (ThermoFisher) cells were used for the amplification of plasmid DNA. Positive clones were selected and cultivated in lysogeny broth (LB) medium at 37 °C for 8 h. E. coli BL21 (DE3) (Agilent) was used for recombinant protein expression (see below).
The cyanobacterium S. elongatus PCC 7942 (Se7942) (Institut Pasteur Paris) was used to obtain genomic DNA of ccmM and ccaA. Se7942 was cultured in BG-11 medium at 30 °C and 50 r.p.m. under continuous light.

A method for GST‐protein purification is described elsewhere 8. Briefly, proteins were expressed in E. coli BL21 (DE3) (Agilent, Santa Clara, CA) or Rosetta (DE3) (Invitrogen) overnight at 18°C and purified by affinity chromatography using GSTrap HP column (GE Healthcare). For the cleavage of the GST tag on LUBEL proteins, dCYLD, OTULIN, vOTU, and E2 enzymes, PreScission Protease (GE Healthcare Life Sciences) was used. A recombinant full‐length LUBEL protein was generated using a baculovirus‐based method via incorporation of the transfer plasmids into the EmBacY bacmid (Geneva Biotech) and subsequent transfection into Sf9 cells. The V0 virus was amplified once to generate a V1 virus stock, which was used to infect larger cultures of Sf9. Expression was performed at 27°C by infecting cells at a density of 2 × 10⁶ cells/ml and harvesting cells 48 h after growth arrest. For the protein purification, Sf9 cell pellets were resuspended in 50 mM HEPES buffer, pH 7.5, 150 mM NaCl, Benzonase, and Complete Protease inhibitors (Roche). Cells were centrifuged for 30 min at 4°C. The soluble anti‐washed (50 mM HEPES, pH 7.5, 150 mM NaCl). LUBEL was eluted (50 mM HEPES, pH 7.5, 150 mM NaCl, 2.5 mM desthiobiotin), and concentrated using a Vivaspin concentrator with a MWCO of 100 kDa. ProTech service was performed by the VBCF Protein Technologies Facility (Vienna, Austria).