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首页> 外文期刊>Biochemistry >Repertoire Selection of Variant Single-Chain Cro:Toward Directed DNA-Bindmg Specificity of Helix-Turn-Helix Proteins
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Repertoire Selection of Variant Single-Chain Cro:Toward Directed DNA-Bindmg Specificity of Helix-Turn-Helix Proteins

机译：变异单链Cro的库选择：螺旋转螺旋蛋白的定向DNA结合特异性。

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摘要

A single-chain derivative of the lambda Cro represser (scCro) has been randomly mutated in amino acid residues critical for specific DNA recognition to create libraries of protein variants.Utilizing phage display-afforded affinity selection,scCro variants have been isolated for binding to synthetic DNA ligands.Isolated scCro variants were analyzed functionally,both in fusion with phage particles and after expression of the corresponding free proteins.The binding properties with regard to specificity and affinity in binding to different DNA ligands were investigated by inhibition studies and determination of equilibrium dissociation constants for formed complexes.Variant proteins with altered DNA-sequence specificity were identified,which favored binding of targeted synthetic DNA sequences over a consensus operator sequence,bound with high affinity by wild-type Cro.The specificities were relatively modest (2-3-fold,as calculated from K_D values),which can be attributed to the inherent properties in the design of the selection system;one half-site of the synthetic DNA sequences maintains the consensus operator sequence,and one "subunit" of the variant single-chain Cro dimers was conserved as wild-type sequence.The anticipated interaction between the wild-type subunit and the consensus DNA half-site of target DNA ligands is,hence,expected to contribute to the overlap in sequence discrimination.The binding affinity for the synthetic DNA sequences,however,was improved 10-30-fold in selected variant proteins as compared to "wild-type" scCro.
机译：λCro阻遏物（scCro）的单链衍生物已在对特定DNA识别至关重要的氨基酸残基中随机突变以创建蛋白质变体文库。 DNA配体。对分离的scCro变体进行功能分析，包括与噬菌体颗粒融合以及表达相应的游离蛋白后进行的功能。通过抑制研究和平衡解离的方法研究了结合特异性和亲和力与不同DNA配体的结合特性。鉴定出具有改变的DNA序列特异性的变体蛋白，该蛋白有利于目标合成DNA序列与共识操纵子序列的结合，并与野生型Cro具有高亲和力。特异性相对适中（2-3-折叠（根据K_D值计算），这可以归因于内在p选择系统的设计;合成DNA序列的一半位点保持共有操纵子序列，变异的单链Cro二聚体的一个“亚基”被保守为野生型序列。因此，预期野生型亚基和靶DNA配体的共有DNA半位点有助于序列识别的重叠。但是，在选定的变体中，对合成DNA序列的结合亲和力提高了10-30倍蛋白质与“野生型” scCro相比。

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