generate Tetra-M2e, we first cloned the tetrameric oligomerization domain of tetrabrachion into the BamHI/BssHII restriction sites of pPEP-T ( Figure 5 ), before cloning complementary oligonucleotides (ATGCATCCCTGGTTCC GCGTGGAAGCCTGCTGACCGAAGTGGAAACCCCGAC-CCGCAACGGCTGGGAATGCAAATGCAGCGATAGCAGC GGATCC) coding for the slightly longer avian M2e sequence (HASLLTEVETPTRNGWECKCSDSSGS) including flanking residues into the N-terminal NsiI/BamHI restriction sites. The M2e-GCN4 construct was made by replacing the nanoparticle fragment of Tetra-M2e with the GCN4 sequence. The plasmids were transformed into Escherichia coli BL21 (DE3) cells, which were grown in Luria broth with ampicillin at 37 • C. Expression was induced with isopropyl β-D-thiogalactopyranoside. Four hours after induction, cells were removed from 37 • C and harvested by centrifugation at 4,000 ×g for 15 min. The cell pellet was stored at −20 • C. The pellet was thawed on ice and suspended in a lysis buffer consisting of 9 M urea, 100 mM NaH 2 PO 4 , 10 mM Tris pH 8, 20 mM imidazole, and 0.2 mM Tris-2-carboxyethl phosphine (TCEP). Cells were lysed by sonication and the lysate was cleared by centrifuging at 30.500 ×g for 45 min. The cleared lysate was incubated with Ni-NTA Agarose Beads (Qiagen, Valencia, CA, USA) for at least 1 hour. The column was washed with lysis buffer and then a buffer containing 9 M urea, 500 mM NaH 2 PO 4 , 10 mM tris pH 8, 20 mM imidazole, and 0.2 mM TCEP. Protein was eluted with a pH gradient: 9 M urea, 100 mM NaH 2 PO 4 , 20 mM citrate, 20 mM imidazole, and 0.2 mM TCEP. Subsequent washes were done at pH 6.3, 5.9, and 4.3. Following the pH gradient, a gradient of lysis buffer with increasing imidazole strength was used to further elute the protein. Purity was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as shown in Figure 6 .