Aslan Filiz M, Yu Yong, Mohr Scott C, Cantor Charles R
Center for Advanced Biotechnology, Department of Chemistry, and Biomolecular Engineering Research Center, Boston University, Boston, MA 02215, USA.
Proc Natl Acad Sci U S A. 2005 Jun 14;102(24):8507-12. doi: 10.1073/pnas.0503112102. Epub 2005 Jun 6.
Streptavidin, a homotetrameric protein with extremely tight biotin binding (K(d) < or = 10(-14) M), has been widely used as an affinity reagent. Its utility would be increased by engineering single-chain mutants with a wide spectrum of affinities, more suitable for phage-display and chip technologies. By a circular permutation procedure, we converted streptavidin to a single-chain dimer (SCD) with two biotin-binding sites and introduced random mutations by error-prone PCR. Clones from a phagemid library, expressed as gene-3 fusion proteins on M13 bacteriophage, were panned with biotinylated beads, and SCD genes from affinity-enriched phage were subcloned to produce soluble proteins. Purification of products from the original gene and two mutants by FPLC and analysis by MALDI-TOF MS showed they exist in both dimeric (single-chain) and tetrameric (two-chain) forms, which were further characterized for their binding affinity to biotin-4-fluorescein (B4F) by fluorescence polarization and intensity measurements. K'(d) values for B4F ranged from approximately 10(-11) to 10(-10) M, although K(d) values for biotin ranged from 10(-6) to 10(-5) M. These results point to the possibility of combining an SCD streptavidin mutant with B4F derivatives to create a fluorescence-tagged affinity system with tight but still-reversible interaction that could be used sequentially with ordinary streptavidin-biotin for composite separation or analysis steps.
链霉亲和素是一种具有极其紧密的生物素结合能力（解离常数K(d)≤10(-14) M）的同四聚体蛋白，已被广泛用作亲和试剂。通过构建具有广泛亲和力谱的单链突变体，其适用性将得到提高，这更适合噬菌体展示和芯片技术。通过环化置换程序，我们将链霉亲和素转化为具有两个生物素结合位点的单链二聚体（SCD），并通过易错PCR引入随机突变。从噬菌粒文库中克隆的基因，在M13噬菌体上作为基因3融合蛋白表达，用生物素化的珠子进行淘选，亲和富集噬菌体的SCD基因被亚克隆以产生可溶性蛋白。通过快速蛋白质液相色谱（FPLC）从原始基因和两个突变体中纯化产物，并通过基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）分析，结果表明它们以二聚体（单链）和四聚体（双链）两种形式存在，通过荧光偏振和强度测量进一步表征它们与生物素-4-荧光素（B4F）的结合亲和力。B4F的表观解离常数K'(d)值范围约为10(-11)至10(-10) M，而生物素的解离常数K(d)值范围为10(-6)至10(-5) M。这些结果表明，有可能将SCD链霉亲和素突变体与B4F衍生物结合，创建一个具有紧密但仍可逆相互作用的荧光标记亲和系统，该系统可与普通链霉亲和素-生物素顺序用于复合分离或分析步骤。
