Department of Chemical and Biological Engineering, University at Buffalo, 905 Furnas Hall, Buffalo, New York 14260, USA.
Biotechnol Bioeng. 2013 Jan;110(1):57-67. doi: 10.1002/bit.24605. Epub 2012 Aug 8.
The coupling between the quaternary structure, stability and function of streptavidin makes it difficult to engineer a stable, high affinity monomer for biotechnology applications. For example, the binding pocket of streptavidin tetramer is comprised of residues from multiple subunits, which cannot be replicated in a single domain protein. However, rhizavidin from Rhizobium etli was recently shown to bind biotin with high affinity as a dimer without the hydrophobic tryptophan lid donated by an adjacent subunit. In particular, the binding site of rhizavidin uses residues from a single subunit to interact with bound biotin. We therefore postulated that replacing the binding site residues of streptavidin monomer with corresponding rhizavidin residues would lead to the design of a high affinity monomer useful for biotechnology applications. Here, we report the construction and characterization of a structural monomer, mSA, which combines the streptavidin and rhizavidin sequences to achieve optimized biophysical properties. First, the biotin affinity of mSA (K(d) = 2.8 nM) is the highest among nontetrameric streptavidin, allowing sensitive monovalent detection of biotinylated ligands. The monomer also has significantly higher stability (T(m) = 59.8 °C) and solubility than all other previously engineered monomers to ensure the molecule remains folded and functional during its application. Using fluorescence correlation spectroscopy, we show that mSA binds biotinylated targets as a monomer. We also show that the molecule can be used as a genetic tag to introduce biotin binding capability to a heterologous protein. For example, recombinantly fusing the monomer to a cell surface receptor allows direct labeling and imaging of transfected cells using biotinylated fluorophores. A stable and functional streptavidin monomer, such as mSA, should be a useful reagent for designing novel detection systems based on monovalent biotin interaction.
链霉亲和素的四级结构、稳定性和功能之间的偶联使其难以构建具有稳定、高亲和力的单体，适用于生物技术应用。例如，链霉亲和素四聚体的结合口袋由来自多个亚基的残基组成，这些残基不能在单个结构域蛋白中复制。然而，最近发现，来自 Rhizobium etli 的根瘤菌亲和素以高亲和力作为二聚体结合生物素，而不需要来自相邻亚基的疏水性色氨酸盖。特别是，根瘤菌亲和素的结合位点使用来自单个亚基的残基与结合的生物素相互作用。因此，我们假设用相应的根瘤菌亲和素残基替换链霉亲和素单体的结合位点残基，将导致设计出适用于生物技术应用的高亲和力单体。在这里，我们报告了结构单体 mSA 的构建和表征，它结合了链霉亲和素和根瘤菌亲和素的序列，以实现优化的生物物理特性。首先，mSA 的生物素亲和力（K(d) = 2.8 nM）是所有非四聚体链霉亲和素中最高的，允许对生物素化配体进行敏感的单价检测。该单体的稳定性（T(m) = 59.8°C）和溶解度也明显高于所有其他以前设计的单体，以确保分子在其应用过程中保持折叠和功能。使用荧光相关光谱法，我们表明 mSA 以单体形式结合生物素化靶标。我们还表明，该分子可用于作为遗传标记，将生物素结合能力引入异源蛋白。例如，将单体重组融合到细胞表面受体上，可以使用生物素化荧光团直接标记和成像转染的细胞。稳定且功能正常的链霉亲和素单体，如 mSA，应该是设计基于单价生物素相互作用的新型检测系统的有用试剂。
