Psectag2 a

The human IL-20 fragment from Leu²⁵ to Glu¹⁷⁶ and mouse IL-20 fragment from Leu²⁵ to Leu¹⁷⁶ was amplified using PCR and inserted into the mammalian expression vector, pSecTag2A (Invitrogen, Carlsbad, Calif.). A Tag consisting of six histidine residues was placed at the C-terminus of the recombinant proteins. The protein was expressed in 293T cells and purified using metal affinity chromatography, as previously described^{19 (link),20 (link)}. The purity of the eluted fractions from the affinity column was checked by the SDS-PAGE test in a reducing condition according to the standard protocol. The purity of human and mouse IL-20 proteins was about 95%.

For protein expression of selected TGM family members, the signal peptide sequence was determined (using the online server SignalP 4.1) and the resulting mature sequence of each (i.e. without signal peptide) was synthesized by GeneArt (Thermo Fisher Scientific, UK) codon-optimised with flanking AscI/NotI restriction digest sites and inserted into a holding vector; codon-optimised nucleotide sequences are presented in Supplementary Table S2. Each family member DNA was subsequently subcloned into mammalian expression vector pSECTag2A (Invitrogen, USA) using restriction sites AscI and NotI, and resultant constructs were sequence verified.
Ten micrograms of purified plasmid DNA of each construct was transiently transfected into HEK293T cells using the calcium chloride method (Promega, USA). Cells were switched into serum-free media (293 SFM II; Gibco, USA) after 24 h, and cultured for up to 4 days, after which point supernatants were collected, filtered through a 0.45 µM filter and dialysed into imidazole-free binding buffer before being loaded onto a 1 ml nickel sulphate charged HiTrap Chelating HP column (GE Healthcare, USA) using a peristaltic pump. Proteins were purified using an Akta Purifier (GE Healthcare) and positive fractions were dialysed into PBS.

The human HATL5 serine protease (SP) domain was cloned into the pSecTag2a plasmid (Life Technologies, Grand Island, NY) for expression and analysis in mammalian cell culture. HATL5 serine protease domain sequence from the human full-length HATL5-V5-His plasmid was PCR amplified using the following primers: 5′-CCAAGGATCCATGTTGTGGGAGACAAGTAGCCAAC-3′ and 5′-CCAAGCGGCCGCGAGTCCAGTCTTGGATGTAATCC-3′. The resulting PCR fragment was cloned into the pSecTag2a vector between the BamHI and NotI sites using standard techniques. Transfection of CHO cells (ATCC, Manassas, VA), protein extraction, and western blot analysis was performed as described above. A mouse monoclonal anti-Myc antibody (Invitrogen, Life Technologies, Grand Island, NY) was used for detection by western blotting.

cDNA of a human FMOD transcript (Genbank assessor number: NM_002023) was subcloned into a commercially available vector pSecTag2A (Life Technology, Grand Island, NY, USA) with C-terminal His-tag, and transfected into CHO-K1 cells (ATCC, Manassas, VA, USA).^{19 (link)} After establishing a stable expression clone, the FMOD was produced and purified by a contract research organization, GenScript (Piscataway, NJ, USA). Briefly, a stable human recombinant FMOD-expressing CHO-K1 cell line was cultured in 1 l serum-free Freestyle CHO Expression Medium (Thermo Fisher Scientific, Canoga Park, CA, USA) at 37 °C with 5% CO₂ in an Erlenmeyer flask. Cell culture supernatant was harvested on day 10 for purification with HiTrap IMAC HP, 1-ml column (GE Healthcare, Uppsala, Sweden). The fractions from a 100 mm imidazole elution were collected and dialyzed against 20 mm PBS, pH 7.4. After that, the sample with low conductivity was loaded onto HiTrapQ HP 1-ml column (GE Healthcare) for further purification. FMOD was then purified under non-reducing conditions, dialyzed again,^{25 (link)} and then subjected to lyophilization. The purity of the FMOD product is 85%. FMOD is reconstituted in PBS, followed by sterilization through a 0.22-μm filter (Thermo Fisher Scientific) before usage.