Structure-based design of a streptavidin mutant specific for an artificial biotin analogue

For a multistep pre-targeting method using antibodies, a streptavidin mutant with low immunogenicity, termed low immunogenic streptavidin mutant No. 314 (LISA-314), was produced previously as a drug delivery tool. However, endogenous biotins (BTNs) with high affinity (Kd < 10(-10) M) for the bind...

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Published in:Journal of biochemistry (Tokyo) 2015-06, Vol.157 (6), p.467-475
Main Authors: Kawato, Tatsuya, Mizohata, Eiichi, Shimizu, Yohei, Meshizuka, Tomohiro, Yamamoto, Tomohiro, Takasu, Noriaki, Matsuoka, Masahiro, Matsumura, Hiroyoshi, Kodama, Tatsuhiko, Kanai, Motomu, Doi, Hirofumi, Inoue, Tsuyoshi, Sugiyama, Akira
Format: Article
Language:English
Subjects:
Biotin - metabolism
Crystallography, X-Ray
Models, Molecular
Mutation
Protein Conformation
Proton Magnetic Resonance Spectroscopy
Streptavidin - chemistry
Streptavidin - genetics
Streptavidin - metabolism
Surface Plasmon Resonance
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Summary:For a multistep pre-targeting method using antibodies, a streptavidin mutant with low immunogenicity, termed low immunogenic streptavidin mutant No. 314 (LISA-314), was produced previously as a drug delivery tool. However, endogenous biotins (BTNs) with high affinity (Kd < 10(-10) M) for the binding pocket of LISA-314 prevents access of exogenous BTN-labelled anticancer drugs. In this study, we improve the binding pocket of LISA-314 to abolish its affinity for endogenous BTN species, therefore ensuring that the newly designed LISA-314 binds only artificial BTN analogue. The replacement of three amino acid residues was performed in two steps to develop a mutant termed V212, which selectively binds to 6-(5-((3aS,4S,6aR)-2-iminohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanoic acid (iminobiotin long tail, IMNtail). Surface plasmon resonance results showed that V212 has a Kd value of 5.9 × 10(-7) M towards IMNtail, but no binding affinity for endogenous BTN species. This V212/IMNtail system will be useful as a novel delivery tool for anticancer therapy.
ISSN:0021-924X
1756-2651
DOI:10.1093/jb/mvv004