Djojonegoro B M, Benedik M J, Willson R C
Department of Biochemical and Biophysical Sciences, University of Houston, Texas 77204.
Biotechnology (N Y). 1994 Feb;12(2):169-72. doi: 10.1038/nbt0294-169.
As a model system for the optimization of separation ligands by bacteriophage surface display, we have constructed a phage surface expression system for a single immunoglobulin-binding domain of Protein A of Staphylococcus aureus. Protein A domain B is genetically fused to the gpIII adsorption protein of the filamentous bacteriophage M13, and hence displayed on the phage surface. Phage displaying the Protein A domain are selectively retained on human IgG-sepharose. Retention is due to specific Protein A-IgG interactions, as demonstrated by competitive inhibition by soluble Protein A or polyclonal human IgG. Polyclonal goat IgG, which is known to bind less well to Protein A than does human IgG, inhibits phage adsorption less effectively. Phage expressing Protein A can be purified in a few rounds of selective adsorption from a vast excess of wild type phage. Diverse libraries constructed by mutagenesis of this construct will allow massive screening of mutant forms of Protein A for alterations in binding and elution properties. We anticipate that phage display will prove to be a widely-applicable method of identification and optimization of affinity ligands for separations.
作为通过噬菌体表面展示优化分离配体的模型系统，我们构建了一种用于金黄色葡萄球菌蛋白A单个免疫球蛋白结合结构域的噬菌体表面表达系统。蛋白A的结构域B与丝状噬菌体M13的gpIII吸附蛋白进行基因融合，从而展示在噬菌体表面。展示蛋白A结构域的噬菌体被选择性地保留在人IgG-琼脂糖上。保留是由于特异性的蛋白A-IgG相互作用，这通过可溶性蛋白A或多克隆人IgG的竞争性抑制得以证明。已知与蛋白A结合不如人IgG紧密的多克隆山羊IgG对噬菌体吸附的抑制效果较差。表达蛋白A的噬菌体可以通过从大量野生型噬菌体中进行几轮选择性吸附来纯化。通过对该构建体进行诱变构建的多样化文库将允许对蛋白A的突变形式进行大规模筛选，以寻找结合和洗脱特性的改变。我们预计噬菌体展示将被证明是一种广泛适用的用于鉴定和优化分离亲和配体的方法。
