Tevsh vector

IL-32θA94V coding sequence was synthesized by Bioneer (Daejeon, Korea) using HT-oligo™ synthesis. The synthesized DNA sequence was cloned into a pTH24 based TEVSH vector (Addgene, Watertown, MA, USA) using NdeI and AgeI restriction sites and rapid DNA ligation kit (Thermo Fisher Scientific, Waltham, MA, USA). TEVSH was a gift from Helena Berglund (Addgene plasmid # 125194; http://n2t.net/addgene:125194; RRID : Addgene 125194) and confirmed by DNA sequencing (Bionics, Seoul, Korea). The recombinant TEVSH vectors were transformed into DH5α by heat shock and purified using a mini prep kit (Intron Biotechnology, Sungnam, Korea). The IL-32θA94V expression vectors were transformed into Rosetta strain of Escherichia coli by heat shock transformation. Successfully transformed single colony was picked up and cultured at 37°C for 4 h in a 200-rpm incubator in Luria–Bertani (LB) media with ampicillin (100 μg/mL). The cultured mixture was transferred to 2.4 L of fresh LB media with ampicillin (100 μg/mL) and grown in a 200-rpm incubator at 37°C until it reached an OD₆₀₀ of 0.6. IL-32θA94V was induced by adding 0.5 mM IPTG. Cells were grown in a 200-rpm incubator at 16°C for 16 h.