pAcGP67A

Soluble recombinant proteins containing the extracellular portion of surface-expressed receptors linked to the Fc region of IgG antibodies help determine ligand specificity for a range of immune system receptors. Among these receptors are the killer cell immunoglobulin-like receptor (KIR) family that recognize HLA class I ligands. These receptors are expressed on natural killer (NK) cells and T cells and play important roles in immune defense and placental development during early pregnancy. Here, researchers describe a method for producing two-domain KIR-Fc fusion proteins using baculovirus-infected insect cells. This study describes the generation of a DNA insert flanked by restriction sites encoding the first 224 amino acids of KIR2D of interest and the Fc region of human IgG1 (Figure 1A). The insert was first cloned into the pAcGP67a transfer vector (Figure 1B). This was subsequently co-transfected into insect cells with linearized baculovirus.

Figure 1. (A) Schematic diagram showing the configuration of a recombinant KIR–Fc fusion gene. The recombinant fusion gene consisting of the D1, D2 and stem domains of a KIR2D molecule (gray box) and the Fc region of a human IgG1 antibody (white box) is cloned using BamH1 and Not1 restriction sites into the multiple cloning site (MCS) of the pAcGP67a vector (B) in frame behind the GP67 secretion signal sequence. Transfection of insect cells produces a soluble recombinant KIR-Fc dimer (C) consisting of the D1, D2, and stem regions of the KIR molecule and the Fc region of the IgG1 antibody. (Hilton H G, et al., 2015)

This approach is more scalable than traditional mammalian cell expression systems and produces efficiently folded proteins that carry post-translational modifications found in native KIRs. The researchers also determined the affinity and specificity of two domains, KIR-Fc, in a multiplex binding assay for a panel of microbeads, each coated with one of 97 HLA class I allotypes. The results obtained from this assay can be used to predict the response of NK cells and T cells when KIR recognizes HLA class I.