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Improvements to the Kunkel mutagenesis protocol for constructing primary and secondary phage-display libraries

机译:用于构建初级和次级噬菌体展示库的Kunkel诱变方案的改进

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摘要

Site-directed mutagenesis is routinely performed in protein engineering experiments. One method, termed Kunkel mutagenesis, is frequently used for constructing libraries of peptide or protein variants in M13 bacteriophage, followed by affinity selection of phage particles. To make this method more efficient, the following two modifications were introduced: culture was incubated at 25°C for phage replication, which yielded 2- to 7-fold more single-stranded DNA template compared to growth at 37°C, and restriction endonuclease recognition sites were used to remove non-recombinants. With both of the improvements, we could construct primary libraries of high complexity and that were 99-100% recombinant. Finally, with a third modification to the standard protocol of Kunkel mutagenesis, two secondary (mutagenic) libraries of a fibronectin type III (FN3) monobody were constructed with DNA segments that were amplified by error-prone and asymmetric PCR. Two advantages of this modification are that it bypasses the lengthy steps of restriction enzyme digestion and ligation, and that the pool of phage clones, recovered after affinity selection, can be used directly to generate a secondary library. Screening one of the two mutagenic libraries yielded variants that bound 2- to 4-fold tighter to human Pak1 kinase than the starting clone. The protocols described in this study should accelerate the discovery of phage-displayed recombinant affinity reagents.
机译:定点诱变通常在蛋白质工程实验中进行。一种被称为“昆克尔诱变”的方法通常用于在M13噬菌体中构建肽或蛋白质变体的文库,然后进行噬菌体颗粒的亲和力选择。为了使该方法更有效,引入了以下两种修饰:将培养物在25°C下孵育以进行噬菌体复制,与在37°C下生长相比,单链DNA模板的产量高出2至7倍,并且限制性内切核酸酶识别位点用于去除非重组子。通过这两项改进,我们可以构建高复杂度的原始文库,并且可以进行99-100%的重组。最后,对Kunkel诱变的标准方案进行第三次修改,构建了纤连蛋白III型(FN3)单体的两个二级(诱变)文库,该文库具有通过易错和不对称PCR扩增的DNA片段。这种修饰的两个优点是它绕开了限制酶消化和连接的冗长步骤,并且亲和力选择后回收的噬菌体克隆库可直接用于生成二级文库。筛选两个诱变文库之一产生的变体与人Pak1激酶的结合比起始克隆紧密2至4倍。这项研究中描述的协议应加快噬菌体展示的重组亲和试剂的发现。

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