Dröge Melloney J, Rüggeberg Carsten J, van der Sloot Almer M, Schimmel Judith, Dijkstra Dolf Swaving, Verhaert Raymond M D, Reetz Manfred T, Quax Wim J
Department of Pharmaceutical Biology, University Centre for Pharmacy, University of Groningen, Antonius Deusinglaan 1, NL-9713 AV Groningen, The Netherlands.
J Biotechnol. 2003 Feb 27;101(1):19-28. doi: 10.1016/s0168-1656(02)00289-4.
Phage display can be used as a protein engineering tool to select proteins with desirable binding properties from a library of randomly constructed mutants. Here, we describe the development of this method for the directed evolution of Bacillus subtilis lipase A, an enzyme that has marked properties for the preparation of pharmaceutically relevant chiral compounds. The lipase gene was cloned upstream of the phage g3p encoding sequence and downstream of a modified g3p signal sequence. Consequently, the enzyme was displayed at the surface of bacteriophage fd as a fusion to its minor coat protein g3p. The phage-bound lipase was correctly folded and fully enzymatically active as determined from the hydrolysis of p-nitrophenylcaprylate with K(m)-values of 0.38 and 0.33 mM for the phage displayed and soluble lipase, respectively. Both soluble lipase and lipase expressed on bacteriophages reacted covalently with a phosphonate suicide inhibitor. The phage does not hamper lipase binding, since both soluble and phage-bound lipase have a similar half-life of inactivation of approximately 5 min. Therefore, we conclude that the Bacillus lipase can be functionally expressed on bacteriophages as a fusion to the phage coat protein g3p. The specific interaction with the suicide inhibitor offers a fast and reproducible method for the future selection of mutant enzymes with an enantioselectivity towards new substrates.
噬菌体展示可作为一种蛋白质工程工具，用于从随机构建的突变体文库中筛选具有理想结合特性的蛋白质。在此，我们描述了这种方法在枯草芽孢杆菌脂肪酶A定向进化中的应用，该酶在制备与药物相关的手性化合物方面具有显著特性。脂肪酶基因被克隆到噬菌体g3p编码序列的上游和一个修饰的g3p信号序列的下游。因此，该酶作为与其次要外壳蛋白g3p的融合蛋白展示在噬菌体fd的表面。通过对硝基苯辛酸酯的水解测定，噬菌体结合的脂肪酶正确折叠且具有完全的酶活性，对于噬菌体展示的脂肪酶和可溶性脂肪酶，其米氏常数（K(m)）分别为0.38和0.33 mM。可溶性脂肪酶和噬菌体上表达的脂肪酶均与膦酸酯自杀抑制剂发生共价反应。噬菌体不会妨碍脂肪酶的结合，因为可溶性脂肪酶和噬菌体结合的脂肪酶具有相似的失活半衰期，约为5分钟。因此，我们得出结论，枯草芽孢杆菌脂肪酶可以作为与噬菌体外壳蛋白g3p的融合蛋白在噬菌体上功能性表达。与自杀抑制剂的特异性相互作用为未来筛选对新底物具有对映选择性的突变酶提供了一种快速且可重复的方法。
