McCafferty J, Jackson R H, Chiswell D J
Cambridge Antibody Technology, Daly Research Laboratories, Babraham, Cambridgeshire, UK.
Protein Eng. 1991 Dec;4(8):955-61. doi: 10.1093/protein/4.8.955.
We have demonstrated that an active enzyme can be expressed on the surface of a bacteriophage. The gene encoding alkaline phosphatase from Escherichia coli was cloned upstream of gene 3, which encodes a minor coat protein of the filamentous bacteriophage, fd. A fusion protein of the correct size was detected from viral particles by Western blotting. Ultrafiltration confirmed that the enzyme fusion behaves as part of a larger structure as would be expected of an enzyme fused to a viral particle. Both wild-type alkaline phosphatase (Arg166) and an active site mutant (Ala166) expressed in this way retain catalytic activity and have qualitatively similar kinetic properties to free enzyme. Values were obtained for Km of 72.7 and 1070 microM respectively whilst relative kcat for the mutant was 36% of that for wild-type. Phage particles expressing alkaline phosphatase were bound to an immobilized inhibitor (arsenate-Sepharose) and eluted with product (20 mM inorganic phosphate). In this way, the functional enzyme is co-purified with the DNA encoding it. This may permit a novel approach to enzyme engineering based on affinity chromatography of mutant enzymes expressed on the phage surface.
我们已经证明，活性酶可以在噬菌体表面表达。将编码大肠杆菌碱性磷酸酶的基因克隆到基因3的上游，基因3编码丝状噬菌体fd的一种次要外壳蛋白。通过蛋白质免疫印迹法从病毒颗粒中检测到了正确大小的融合蛋白。超滤证实，酶融合体表现为更大结构的一部分，这与融合到病毒颗粒上的酶的预期情况相符。以这种方式表达的野生型碱性磷酸酶（Arg166）和活性位点突变体（Ala166）均保留催化活性，并且在动力学性质上与游离酶定性相似。分别测得野生型和突变体的米氏常数（Km）值为72.7和1070微摩尔，而突变体的相对催化常数（kcat）为野生型的36%。表达碱性磷酸酶的噬菌体颗粒与固定化抑制剂（砷酸 - 琼脂糖）结合，并用产物（20 mM无机磷酸盐）洗脱。通过这种方式，功能性酶与其编码DNA一起被共纯化。这可能为基于噬菌体表面表达的突变酶亲和层析的酶工程提供一种新方法。
