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首页> 外文期刊>The FEBS journal >In vitro selection and characterization of a stable subdomain of phosphoribosylanthranilate isomerase
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In vitro selection and characterization of a stable subdomain of phosphoribosylanthranilate isomerase

机译：核糖二氰基氨基苯甲酸异核酶稳定亚域的体外选择和表征

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The (beta alpha)(8)-barrel is the most common enzyme fold and it is capable of catalyzing an enormous diversity of reactions. It follows that this scaffold should be an ideal starting point for engineering novel enzymes by directed evolution. However, experiments to date have utilized in vivo screens or selections and the compatibility of (beta alpha)(8)-barrels with in vitro selection methods remains largely untested. We have investigated plasmid display as a suitable in vitro format by engineering a variant of phosphoribosylanthranilate isomerase (PRAI) that carried the FLAG epitope in active-site-forming loop 6. Trial enrichments for binding of mAb M2 (a mAb to FLAG) demonstrated that FLAG-PRAI could be identified from a 10(6)-fold excess of a FLAG-negative competitor in three rounds of in vitro selection. These results suggest PRAI as a useful scaffold for epitope and peptide grafting experiments. Further, we constructed a FLAG-PRAI loop library of approximate to 10(7) clones, in which the epitope residues most critical for binding mAb M2 were randomized. Four rounds of selection for antibody binding identified and enriched for a variant in which a single nucleotide insertion produced a truncated (beta alpha)(8)-barrel consisting of (beta alpha)(1-5)beta(6). Biophysical characterization of this clone, trPRAI, demonstrated that it was selected because of a 21-fold increase in mAb M2 affinity compared with full-length FLAG-PRAI. Remarkably, this truncated barrel was found to be soluble, structured, thermostable and monomeric, implying that it represents a genuine subdomain of PRAI and providing further evidence that such subdomains have played an important role in the evolution of the (beta alpha)(8)-barrel fold.
机译：β-α（8）-桶是最常见的酶折叠，它能够催化大量的反应。因此，该支架应该是通过定向进化工程化新酶的理想起点。然而，迄今为止的实验已经利用了体内筛选或选择，并且β-α（8）-桶与体外选择方法的相容性仍未经测试。我们已经通过工程化在活性位点形成环6中带有FLAG表位的磷酸核糖基邻氨基苯甲酸酯异构酶（PRAI）的变体，研究了质粒显示作为合适的体外形式的方法。在三轮体外选择中，FLAG-PRAI可以从FLAG阴性竞争对手的10（6）倍过量中鉴定出来。这些结果表明PRAI是用于表位和肽移植实验的有用支架。此外，我们构建了大约10（7）个克隆的FLAG-PRAI循环文库，其中对结合mAb M2最关键的表位残基被随机化。针对抗体结合的四轮选择进行了鉴定，并针对其中单个核苷酸插入产生了由（βα）（1-5）β（6）组成的截短的（βα）（8）桶的变体进行了富集。该克隆trPRAI的生物物理特性表明，之所以选择该克隆是因为与全长FLAG-PRAI相比，mAb M2亲和力增加了21倍。值得注意的是，该截短的桶被发现是可溶的，结构化的，热稳定的和单体的，这意味着它代表了PRAI的真正亚域，并提供了进一步的证据表明这些亚域在（beta alpha）的进化中发挥了重要作用（8） -桶折。

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