Soumillion P, Jespers L, Bouchet M, Marchand-Brynaert J, Sartiaux P, Fastrez J
Laboratoire de Biochimie Physique et des Biopolyméres, Université Catholique de Louvain, Belgium.
Appl Biochem Biotechnol. 1994 May-Jun;47(2-3):175-89; discussion 189-90. doi: 10.1007/BF02787933.
Despite recent progress, our understanding of enzymes remains limited: the prediction of the changes that should be introduced to alter their properties or catalytic activities in an expected direction remains difficult. An alternative to rational design is selection of mutants endowed with the anticipated properties from a large collection of possible solutions generated by random mutagenesis. We describe here a new technique of in vitro selection of genes on the basis of the catalytic activity of the encoded enzymes. The gene coding for the enzyme to be engineered is cloned into the genome of a filamentous phage, whereas the enzyme itself is displayed on its surface, creating a phage enzyme. A bifunctional organic label containing a suicide inhibitor of the enzyme and a ligand with high affinity for an immobilized receptor are constructed. On incubation of a mixture of phage enzymes, those phages showing an activity on the inhibitor under the conditions of the experiment are labeled. These phages can be recovered by affinity chromatography. The design of the label and the factors controlling the selectivity of the selection are analyzed. The advantages of the technique and its scope in terms of the enzymes that can be engineered are discussed.
尽管最近取得了进展，但我们对酶的理解仍然有限：预测为使酶的性质或催化活性朝着预期方向改变而应引入的变化仍然很困难。理性设计的替代方法是从通过随机诱变产生的大量可能解决方案中选择具有预期性质的突变体。我们在此描述一种基于编码酶的催化活性进行体外基因选择的新技术。将待改造酶的编码基因克隆到丝状噬菌体的基因组中，而酶本身则展示在其表面，从而产生噬菌体酶。构建一种双功能有机标记物，其包含该酶的自杀抑制剂和对固定化受体具有高亲和力的配体。在噬菌体酶混合物孵育时，那些在实验条件下对抑制剂有活性的噬菌体被标记。这些噬菌体可通过亲和色谱法回收。分析了标记物的设计以及控制选择选择性的因素。讨论了该技术的优点及其在可改造酶方面的应用范围。
