Figure 4.

Examples of designer microgenes. (A) Sequence of 14 amino acids was extracted from the anti-paralleled coiled coil α-helix region of the N-terminal domain of Escherichia coli seryl-tRNA synthetase (30). The structure of the enzyme from T. thermophilus is shown (29), and the corresponding region in this enzyme is represented in blue [drawn using Rasmol (31)]. (B) From the extracted sequence, a microgene was designed so that one of six frames codes for the extracted sequence and the others do not contain any termination codons. The sixth residue (Val) in the extracted sequence had to be changed to Glu in the microgene to avoid the appearance of a termination codon in other frames. (C) MPR primers were designed for the microgene. Mismatched nucleotides were added at 3′-OH ends of both primers (in green). (D) An example of a microgene polymer (pYT071) made from KY-859 and KY-860. MPR was carried out for 65 cycles using Vent (exo⁺) DNA polymerase. The annealing and extension temperature was 63°C. Inserted nucleotides at junctions are in red. (E–H) Design of a microgene from a parallel β-helix protein, E. chrysanthemi pectate lyase E (32), and an example of a microgene polymer. The strategy was same as for A–D, except the 22-amino acid sequence was a consensus sequence comprised of three β-strands.