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    Structure-based design of a streptavidin mutant specific for an artificial biotin analogue.
    J Biochem 2015 Jun 2;157(6):467-75. Epub 2015 Feb 2.
    Division of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan; Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo 153-8904, Japan; and Radioisotope Center, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
    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.

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    Crystal structure of streptavidin mutant with low immunogenicity.
    J Biosci Bioeng 2015 Jun 26;119(6):642-7. Epub 2014 Nov 26.
    Radioisotope Center, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan. Electronic address:
    We previously created a low-immunogenic core streptavidin mutant No. 314 (LISA-314) by replacing six amino-acid residues for use as a delivery tool for an antibody multistep pre-targeting process (Yumura et al., Protein Sci. Read More
    Structure-based design and synthesis of a bivalent iminobiotin analog showing strong affinity toward a low immunogenic streptavidin mutant.
    Biosci Biotechnol Biochem 2015 3;79(4):640-2. Epub 2015 Jan 3.
    a Division of Applied Chemistry, Graduate School of Engineering , Osaka University , Suita , Japan.
    The streptavidin/biotin interaction has been widely used as a useful tool in research fields. For application to a pre-targeting system, we previously developed a streptavidin mutant that binds to an iminobiotin analog while abolishing affinity for natural biocytin. Here, we design a bivalent iminobiotin analog that shows 1000-fold higher affinity than before, and determine its crystal structure complexed with the mutant protein. Read More
    Evolved streptavidin mutants reveal key role of loop residue in high-affinity binding.
    Protein Sci 2011 Jul 12;20(7):1145-54. Epub 2011 May 12.
    Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
    We have performed a detailed analysis of streptavidin variants with altered specificity towards desthiobiotin. In addition to changes in key residues which widen the ligand binding pocket and accommodate the more structurally flexible desthiobiotin, the data revealed the role of a key, non-active site mutation at the base of the flexible loop (S52G) which slows dissociation of this ligand by approximately sevenfold. Our data suggest that this mutation results in the loss of a stabilizing contact which keeps this loop open and accessible in the absence of ligand. Read More
    A streptavidin mutant with altered ligand-binding specificity.
    Proc Natl Acad Sci U S A 1998 Nov;95(23):13525-30
    Center for Advanced Biotechnology and Departments of Physics, Biomedical Engineering, and Pharmacology and Experimental Therapeutics, Boston University, Boston, MA 02215, USA.
    The biotin-binding site of streptavidin was modified to alter its ligand-binding specificity. In natural streptavidin, the side chains of N23 and S27 make two of the three hydrogen bonds with the ureido oxygen of biotin. These two residues were mutated to severely weaken biotin binding while attempting to maintain the affinity for two biotin analogs, 2-iminobiotin and diaminobiotin. Read More