Fig. 1. Phage selection and optimization of best Ni²⁺ cleavage sequence.

a. Substrate phage construct. b. Sequence frequency plot of Ni²⁺ cleavage selection, red dashed line indicates cleavage site between P₁ and P₁’ positions, P₂’ to P₇’ correspond to the randomized positions in Fig. 1a. Panels c-e. Evaluation of the cleavage of synthetic peptides (black arrow indicates uncleaved peptide). c. The most frequently observed phage selected peptide sequence YFLGGSHHTDLPGGSRRLFY; d. optimized peptide YFLPGSRHWG; e. best optimized peptide YFLPGSHHWG (the Arg for His substitution is based on the sequence logo in Fig. 1b). All peptides contain C-terminal carboxamides. The cleavage conditions were: peptide 0.2 mM, 0.1 M CHES, pH 8.2, 1 mM NiCl₂, 22 °C, 16 hours. The mass of uncleaved and cleaved peptides were measured using MALDI-TOF shown in Supplementary Fig. 5.