Selection of a platinum-binding sequence in a loop of a four-helix bundle protein
Section snippets
Construction of the phage display LARFH library
To create an LARFH gene library, amino acid residues at positions 21, 22, 23, 25, and 26 were subjected to semi-random mutagenesis (Fig. 1). Each of their codons was replaced with the NVS codon (N = A + T + G + C, V = A + G + C, S = G + C). Genes were then amplified by using two different primers so that the EcoRI and HindIII recognition sites could be added at the 5′ and 3′ termini, respectively. Amplified DNA fragments were digested with EcoRI and HindIII and then ligated to T7 Select Vector
Selection of mutant LARFH that interacts with platinum
Fig. 1A shows the amino acid sequence of LARFH, with rectangles representing segments that are likely to form α-helices. We inserted a platinum-binding sequence in the loop connecting the first and second α-helices. When LARFH is displayed on phage particles, the first loop may be positioned at the opposite face of the phage and may be well exposed to solvent (Fig. 1B). Therefore, we considered that platinum and other metal particles might access this loop more easily. The residues Ser21,
Discussion
In this study, we successfully inserted a high-affinity platinum binding site in LARFH, which enabled the protein to site-specifically bind to a platinum surface. The mutant protein with an YKRGYK sequence in its first loop seems to bind to platinum more strongly than wild-type LARFH. The KD of mutant LARFH binding to platinum is 1.0 × 10−8 M, which is better than those of peptides whose binding constants have been previously measured (21); for example, 3l-PtBP2 has a reported Keq of 6.7 × 106 M
Acknowledgments
The work was supported by MEXT-Supported Program for the Strategic Research Foundation at Private Universities (S1512002), 2015–2017 and by JSPS KAKENHI Grant Number 16K14494.
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