Cloning and Expression of CD58 and CD2

The coding sequences for the ORF or the extracellular domain of cd58 and cd2 were amplified through RT-PCR by using primers (shown in Table S1 in Supplementary Material) containing an EcoRI site added to the 5′ end and an XhoI site added to the 3′ end. The PCR products were digested and ligated into pEGFP-N1 (Clontech) or pcDNA6/myc-His©B (Invitrogen) to construct eukaryotic expression vectors (pEGFP-cd58, pEGFP-cd2, and pcDNA6-cd58) with enhanced GFP-tag or myc-tag and into pMalc2e to construct prokaryotic expression vectors (pMalc2e-cd2) with MBP-tag (43 (link)). For eukaryotic expression of Cd58 protein, the plasmid DNA was transformed into HEK293T cells. For prokaryotic expression of Cd2 protein, the pMalc2e-cd2 was transformed into E. coli Rosetta (DE3) pLysS. Positive colonies were inoculated into Luria–Bertani medium containing kanamycin (50 µg/mL) and the protein expression was induced by isopropyl-β-d-thio-galactoside (1 mM/mL) as previously described (31 (link)). The recombinant proteins were detected via SDS-PAGE and purified through Amylose resin affinity chromatography in accordance with the manufacturer’s manual (NEB, pMAL system).