Pacgp67

The Ig1-3-FNIII12 domain (aa 27A–513A), Ig1-3-A⁺B⁺ (aa 30D-318L), and FN12 (aa 312Q–510G) of human LAR were cloned into pAcGP67 (BD Bioscience), modified for C-terminal protein-A tagging. Since human SALM5 was hardly expressed in High Five insect cells (Invitrogen), hybrid LRR techniques were used to generate a chimeric hySALM5 proteins67 (link). Briefly, the N terminus of VLRB61 (1M–82L) was combined with the LRR and Ig domains of human SALM5 (aa 59A–374I) by overlap PCR, and the resulting chimeric gene for hySALM5 was cloned into pVL1393 (Invitrogen), modified for C-terminal Fc tagging. All constructs described above contain a thrombin cleavage site (LVPRGS) between target proteins and tags. The High Five insect cells were transfected with corresponding P4 baculovirus for 3 days and harvested. The supernatants containing secreted proteins were loaded onto IgG sepharose column to purify protein A-fused proteins, or onto protein A sepharose column to purify Fc-tagged protein. The protein A or Fc tags were thrombin cleaved (0.5% v/v) for 16 hours at 4 °C, followed by gel-filtration purification using Superdex 200 (GE Healthcate Life Science).