Directed evolution of PDZ variants to generate high-affinity detection reagents
Directed evolution of PDZ variants to generate high-affinity detection reagents
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DOI：
10.1093/protein/gzi018
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发表时间：
2005-04-01
影响因子：
2.4
通讯作者：
Inglese, J
中科院分区：
文献类型：
--
作者：
Ferrer, M;Maiolo, J;Inglese, J
高通量的蛋白水解酶分析被用来鉴定有潜力成为有价值的治疗产品的新的蛋白水解酶抑制剂。在蛋白水解酶分析中，抗体作为亲和试剂检测蛋白水解物是非常有用的，但分离和生产这种抗体是不可靠的、缓慢的和昂贵的。先前已经表明，PDZ结构域也可以用于检测高通量均相分析中的蛋白水解物，但其有限的天然谱系限制了其仅用于少数几个肽。在这里，我们表明定向进化是一种有效的方法来创建新的PDZ结构域来检测蛋白酶活性。我们报告了第一次使用噬菌体展示来改变PDZ结构域的特异性，产生了三个变异体，与HIV蛋白酶的多肽裂解产物的亲和力增加了25倍。在NHERF PDZ结构域的一些变异体中发现的氨基酸替换被分配了三个不同的角色：与配体残基-1的特异性‘β1-β3’相互作用，与配体残基-4至-7的相互作用，以及噬菌体展示效率的提高。这些变种具有高达620 nm的亲和力，在需要较少试剂的情况下，检测灵敏度提高了5倍以上。这里展示的方法导致了用于药物发现的高灵敏试剂的方法，这种试剂可以更可靠地分离并以更低的成本生产。
High-throughput protease assays are used to identify new protease inhibitors which have the potential to become valuable therapeutic products. Antibodies are of great utility as affinity reagents to detect proteolysis products in protease assays, but isolating and producing such antibodies is unreliable, slow and costly. It has been shown previously that PDZ domains can also be used to detect proteolysis products in high-throughput homogeneous assays but their limited natural repertoire restricts their use to only a few peptides. Here we show that directed evolution is an efficient way to create new PDZ domains for detection of protease activity. We report the first use of phage display to alter the specificity of a PDZ domain, yielding three variants with up to 25-fold increased affinity for a peptide cleavage product of HIV protease. Three distinct roles are assigned to the amino acid substitutions found in the selected variants of the NHERF PDZ domain: specific 'beta 1-beta 3' interaction with ligand residue -1, interactions with ligand residues -4 to -7 and improvement in phage display efficiency. The variants, having affinities as high as 620 nM, display improvements in assay sensitivity of over 5-fold while requiring smaller amounts of reagents. The approach demonstrated here leads the way to highly sensitive reagents for drug discovery that can be isolated more reliably and produced less expensively.