Affinity maturation of a Taq DNA polymerase specific affibody by helix shuffling
Affinity maturation of a Taq DNA polymerase specific affibody by helix shuffling
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DOI：
10.1093/protein/12.10.873
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发表时间：
1999-10-01
期刊：
影响因子：
--
通讯作者：
Nygren, PÅ
中科院分区：
文献类型：
--
作者：
Gunneriusson, E;Nord, K;Nygren, PÅ
通过α-螺旋改组策略研究了提高标签DNA聚合酶特异性结合蛋白(亲和体)亲和力的可能性。初步的亲和体来自葡萄球菌蛋白A的三螺旋Z结构域的一个朴素的组合文库，通过选择性地重新随机化该结构域的两个α-螺旋之一的六个氨基酸位置而构建了一个分级文库，组成Tag DNA聚合酶结合表面，经单价噬菌体展示技术筛选后，通过生物传感器技术确定了与Tag DNA聚合酶的亲和力(K-D)在30-50 nM范围内的第二代变异体，结合数据分析表明亲和力的增加主要是由于：解离速率动力学降低。有趣的是，观察到的第二代标签DNA的亲和力。聚合酶特异的亲和抗体具有与人免疫球蛋白G的原始蛋白A结构域和Fc结构域之间的亲和力相似的强度。此外，通过头尾基因融合构建的第二代亲和抗体的二聚体版本证明了通过亲和抗体的多聚体增加表观亲和力的可能性。与其单体相比，传感器芯片固定的Tag DNA聚合酶结合的表观亲和力比其单体高三倍，这是由于减速动力学的原因。结果表明，蛋白A结构域的结合特异性可以重定向到完全不同的靶点，而不会损失结合强度。
The possibility of increasing the affinity of a Tag DNA polymerase specific binding protein (affibody) was investigated by an alpha-helix shuffling strategy. The primary affibody was from a naive combinatorial library of the three-helix bundle Z domain derived from staphylococcal protein A, A hierarchical library was constructed through selective re-randomization of six amino acid positions in one of the two alpha-helices of the domain, making up the Tag DNA polymerase binding surface, After selections using monovalent phage display technology, second generation variants were identified having affinities (K-D) for Tag DNA polymerase in the range of 30-50 nM as determined by biosensor technology, Analysis of binding data indicated that the increases in affinity were predominantly due to:decreased dissociation rate kinetics. Interestingly, the affinities observed for the second generation Tag DNA. polymerase specific affibodies are of similar strength as the affinity between the original protein A domain and the Fc domain of human immunoglobulin G. Further, the possibilities of increasing the apparent affinity through multimerization of affibodies was demonstrated for a dimeric version of one of the second generation affibodies, constructed by head-to-tail gene fusion. As compared with its monomeric counterpart, the binding to sensor chip immobilized Tag DNA polymerase was characterized by a threefold higher apparent affinity, due to slower off-rate kinetics. The results show that the binding specificity of the protein A domain can be re-directed to an entirely different target, without loss of binding strength.