Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada.
PLoS One. 2013 Jul 16;8(7):e69530. doi: 10.1371/journal.pone.0069530. Print 2013.
Development of a high-affinity streptavidin-binding peptide (SBP) tag allows the tagged recombinant proteins to be affinity purified using the streptavidin matrix without the need of biotinylation. The major limitation of this powerful technology is the requirement to use biotin to elute the SBP-tagged proteins from the streptavidin matrix. Tight biotin binding by streptavidin essentially allows the matrix to be used only once. To address this problem, differences in interactions of biotin and SBP with streptavidin were explored. Loop3-4 which serves as a mobile lid for the biotin binding pocket in streptavidin is in the closed state with biotin binding. In contrast, this loop is in the open state with SBP binding. Replacement of glycine-48 with a bulkier residue (threonine) in this loop selectively reduces the biotin binding affinity (Kd) from 4 × 10(-14) M to 4.45 × 10(-10) M without affecting the SBP binding affinity. Introduction of a second mutation (S27A) to the first mutein (G48T) results in the development of a novel engineered streptavidin SAVSBPM18 which could be recombinantly produced in the functional form from Bacillus subtilis via secretion. To form an intact binding pocket for tight binding of SBP, two diagonally oriented subunits in a tetrameric streptavidin are required. It is vital for SAVSBPM18 to be stably in the tetrameric state in solution. This was confirmed using an HPLC/Laser light scattering system. SAVSBPM18 retains high binding affinity to SBP but has reversible biotin binding capability. The SAVSBPM18 matrix can be applied to affinity purify SBP-tagged proteins or biotinylated molecules to homogeneity with high recovery in a reusable manner. A mild washing step is sufficient to regenerate the matrix which can be reused for multiple rounds. Other applications including development of automated protein purification systems, lab-on-a-chip micro-devices, reusable biosensors, bioreactors and microarrays, and strippable detection agents for various blots are possible.
开发高亲和力链霉亲和素结合肽 (SBP) 标签可使标记的重组蛋白无需生物素化即可使用链霉亲和素基质进行亲和纯化。这项强大技术的主要限制是需要使用生物素来从链霉亲和素基质中洗脱 SBP 标记的蛋白质。链霉亲和素对生物素的紧密结合基本上使得该基质只能使用一次。为了解决这个问题，我们探索了生物素和 SBP 与链霉亲和素相互作用的差异。Loop3-4 作为链霉亲和素中生物素结合口袋的可移动盖，在与生物素结合时处于关闭状态。相比之下，当与 SBP 结合时，该环处于打开状态。用较大的残基（苏氨酸）替换该环中的甘氨酸-48，可选择性地将生物素结合亲和力 (Kd) 从 4×10(-14) M 降低至 4.45×10(-10) M，而不影响 SBP 结合亲和力。在第一个突变体 (G48T) 中引入第二个突变 (S27A)，可产生一种新型工程化链霉亲和素 SAVSBPM18，该亲和素可通过分泌从枯草芽孢杆菌中以功能形式重组产生。为了形成用于 SBP 紧密结合的完整结合口袋，四聚体中的两个对角定向亚基是必需的。对于 SAVSBPM18 来说，在溶液中稳定处于四聚体状态至关重要。这一点通过高效液相色谱/激光散射系统得到了证实。SAVSBPM18 保持对 SBP 的高结合亲和力，但具有可逆的生物素结合能力。SAVSBPM18 基质可用于以可重复使用的方式高度纯化 SBP 标记的蛋白质或生物素化分子。温和的洗涤步骤足以使基质再生，可重复使用多轮。其他应用包括开发自动化蛋白质纯化系统、芯片实验室微器件、可重复使用的生物传感器、生物反应器和微阵列，以及用于各种印迹的可剥离检测试剂。
