Soumillion P, Jespers L, Bouchet M, Marchand-Brynaert J, Winter G, Fastrez J
Laboratoire de Biochimie Physique et des Biopolymères, Université Catholique de Louvain, Belgium.
J Mol Biol. 1994 Apr 8;237(4):415-22. doi: 10.1006/jmbi.1994.1244.
Recently the display of repertoires of peptides and proteins on the surface of filamentous phage, and selection of the phage by binding to a ligand, has allowed the isolation of peptides and proteins with rare binding activities. Furthermore, phages displaying enzymes (phage enzymes) have been selected by affinity of binding to inhibitors. Here we show, using a suicide inhibitor, that phage enzymes can also be selected by their catalytic activity. Two phage enzymes were constructed by fusion to the minor coat protein of the phage (g3p), displaying either an active beta-lactamase or a catalytically inactive mutant in which the essential serine of the active site was mutated to alanine. The phages were then incubated with a beta-lactamase suicide inhibitor connected by a spacer to a biotin moiety. The active (but not the inactive) phages were labelled, and the active phages selected from mixtures with inactive phages by binding and elution from streptavidin-coated beads. The selection ratio for active versus inactive phages (about ten on elution of the phages by reduction of an S-S bond in the spacer between the warhead and biotin) could be improved to about 50 on elution by proteolytic cleavage of beta-lactamase from g3p at an intervening factor X site. Selection of phage-enzymes by catalysis may provide a means of creating new enzymes and refining their catalytic properties.
最近，丝状噬菌体表面肽和蛋白质文库的展示，以及通过与配体结合对噬菌体进行筛选，使得具有罕见结合活性的肽和蛋白质得以分离。此外，通过与抑制剂的结合亲和力筛选出了展示酶的噬菌体（噬菌体酶）。在此我们表明，使用自杀性抑制剂，噬菌体酶也可以通过其催化活性进行筛选。通过与噬菌体的次要外壳蛋白（g3p）融合构建了两种噬菌体酶，分别展示有活性的β-内酰胺酶或催化无活性的突变体，其中活性位点的必需丝氨酸被突变为丙氨酸。然后将噬菌体与通过间隔物连接到生物素部分的β-内酰胺酶自杀性抑制剂一起孵育。活性（而非无活性）噬菌体被标记，通过与链霉亲和素包被的珠子结合和洗脱，从与无活性噬菌体的混合物中筛选出活性噬菌体。活性噬菌体与无活性噬菌体的筛选比例（在通过还原弹头与生物素之间间隔物中的二硫键洗脱噬菌体时约为10），在通过中间的因子X位点将β-内酰胺酶从g3p上进行蛋白水解切割洗脱时可提高到约50。通过催化作用筛选噬菌体酶可能提供一种创造新酶并优化其催化特性的方法。
