Fig. 1. (a) Structure of FRET-PD-1: AA position 123–140 was selected to make the FRET-PD-1 peptide substrate in which MCA (4-methyl-coumaryl-7-amide; fluorescent reagent) was added at the N-terminal and DNP (2,4-dinitrophenyl; quenching reagent) at the C-terminal. Lysine was inserted in order to bind the DNP. (b) Results of screening tests: of 45 clones investigated, the H34 light chain had the highest activity for cleaving the FRET-PD-1 substrate. Light chain: 5 μM, FRET-PD-1: 25 μM, buffer: PBS (pH 7.4), reaction temperature: 37 °C. (c) Amino acid sequences: the amino acid (aa) sequences of the Vk region (1–95) of germline Vk 1D-39-01 (major clone belonging to subgroup I), and variable regions (1–107) of H34 and H34-Pro⁹⁵(+) are presented. Pro⁹⁵ is highly conserved in subgroup I. It is characteristic that Pro⁹⁵ was lacking in H34.