Construction of artificial proteins and in vitro antigen presentation assay.

(A) Amino acid sequences encoded by the microgenes MG2101 and MG6101. Three different reading frames of each microgene encoded three peptides with different amino acid sequences (MG-2101 and MG6101 and Figure S1B, Supporting Information). MHC class I epitope (OVA-I) and class II epitope (OVA-II) are shown in red and blue, respectively. Sequences forming α-helical and β-sheet structures are shown in sky blue and brown, respectively. (B) Scheme for generating artificial proteins. Designer microgenes (MG-2101 and MG6101) were polymerized using the microgene polymerization reaction (MPR) [8] with primers designed from the microgene sequences (Figure S1B). These primers contain complementary bases in their 3′ regions and a mismatched base at their 3′-OH ends, which enables efficient polymerization of the microgene. Thermal cycling reactions with MPR primers, the four dNTPs and thermostable exo+ DNA polymerase yields head-to-tail polymers of the microgenes. One intriguing feature of MPR is that the reaction randomly inserts or deletes nucleotides at end-joining junctions, thereby changing the reading frame of the polymers [8]. (C) Schematic drawing of the eight artificial proteins used in the initial screening; the amino acid sequences of these proteins are shown in Figure S2, Supporting Information. (D) Comparison of the abilities of artificial antigens and native OVA to induce antigen presentation in vitro. OVA-specific T-cells were cultured with dendritic cells in the presence of an antigen. The level of IL-2 in the culture supernatant was determined by ELISA. Data shown are mean IL-2 concentration ± standard deviations (SD) (n = 3). Only F182A showed a positive reaction. MT290, MT297 and MT332 are artificial proteins without either OVA-I or OVA-II. Inset: SDS-PAGE of purified artificial proteins.