Pfuse migg2a fc1 vector

Genes encoding the human or mouse forms of TEM8 fused to the human Fc protein were cloned into the pFUSE-mIgG2A-Fc1 vector (InvivoGen, San Diego, CA). TEM8 protein production was then carried out with the help of the Protein Expression Facility, University of Birmingham. Human TEM8-Fc/pFUSE-mIgG2A or mouse TEM8-Fc/pFUSE-mIgG2A plasmids were transfected into 293T cells using polyethylenimine (PEI 25000; Polysciences, Warrington, PA) in Opti-MEM Reduced Serum Medium (Gibco) and 7 days later supernatants were harvested and protein purified using Protein A Sepharose CL-4B (GE Healthcare) according to the manufacturer's instructions.

A pCMV6-AC expression vector containing the MICA gene (Origene, Rockville, ML, USA) was altered by site-directed mutagenesis at two positions (codons for amino acids 24 and 360) to obtain the MICA*00701 allele. This allele has a methionine at amino acid position 129. The MICA-129Val variant (pCMV6-AC-MICA-129Val) was generated by mutagenesis of the pCMV6-AC-MICA-129Met construct. To obtain MICA proteins as mouse IgG_2a-Fc fusion proteins (MICA-129Met-mIgG_2a-Fc and MICA-129Val-mIgG_2a-Fc), the extracellular parts of MICA were amplified by polymerase chain reaction (PCR) from pCMV6-AC-MICA-129Met and pCMV6-AC-MICA-129Val vectors. As control, an ovalbumin (OVA) fusion protein construct (OVA-mIgG_2a-Fc) was generated. Primers with restriction sites for KpnI (MICA-129-Fc_fwd: 5′-TGGTACCATGGGGCTGGGCCCGGTCTTCCTGC-3′; OVA-Fc_fwd: 5′-CAGGTACCATGGGCTCCATCGGCGCAGCAA-3′) or BamHI (MICA-129-Fc_rev: 5′-CGGATCCTGAAGCACCAGCACTTTCCCAGA-3′; OVA-Fc_rev: 5′-TGAGGATCCATAGGGGAAACACATCTGCCAAAG-3′) were used, and the PCR products were inserted into the pcDNA3.1/myc-His A(+) expression vector (Invitrogen, Darmstadt, Germany) that already contained the mouse IgG_2a-Fc cDNA, including the hinge and the CH2 and CH3 regions, derived from the pFUSE-mIgG_2a-Fc1 vector (InvivoGen, Toulouse, France). All constructs were sequenced before use.