Circularly permuted proteins (cpPs) represent a novel type of mutant proteins with original termini that are covalently linked through a peptide connector and opened at any other place of the polypeptide backbone to create new ends. cpPs are finding wide applications in biotechnology because their properties may be quite different from those of the parental protein. However, the actual challenge for the creation of successful cpPs is to identify those peptide bonds that can be broken to create new termini and ensure functional and well-folded cpPs. Herein, we describe CiPerGenesis, a combinatorial mutagenesis approach that uses two oligonucleotide libraries to amplify a circularized gene by PCR, starting and ending from a focused target region. This approach creates small libraries of circularly permuted genes that are easily cloned in the correct direction and frame using two different restriction sites encoded in the oligonucleotides. Once expressed, the protein libraries exhibit a unique sequence diversity, comprising cpPs that exhibit ordinary breakpoints between adjacent amino acids localized at the target region as well as cpPs with new termini containing user-defined truncations and repeats of some amino acids. CiPerGenesis was tested at the lid region G134–H148 of green fluorescent protein (GFP), revealing that the most fluorescent variants were those starting at Leu141 and ending at amino acids Tyr145, Tyr143, Glu142, Leu141, Lys140, and H139. Purification and biochemical characterization of some variants suggested a differential expression, solubility and maturation extent of the mutant proteins as the likely cause for the variability in fluorescence intensity observed in colonies.

中文翻译：

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CiPerGenesis，一种诱变方法，可产生在特定区域随机开放的圆形排列蛋白的小文库：绿色荧光蛋白的测试

环状排列的蛋白质（cpP）代表一种新型的具有原始末端的突变蛋白质，该末端蛋白质通过肽连接器共价连接，并在多肽骨架的任何其他位置打开以产生新的末端。cpP在生物技术中发现了广泛的应用，因为它们的性质可能与亲本蛋白质的性质完全不同。但是，创建成功的cpP的实际挑战是识别可以被断裂以创建新末端的肽键，并确保功能性和折叠良好的cpP。在本文中，我们描述了CiPerGenesis，这是一种组合诱变方法，它使用两个寡核苷酸文库通过PCR扩增环化基因，从聚焦目标区域开始和结束。这种方法创建了圆形排列的基因的小文库，使用寡核苷酸中编码的两个不同的限制性酶切位点，可以容易地以正确的方向和框架克隆它们。蛋白质文库一旦表达，便表现出独特的序列多样性，包括在目标区域附近的相邻氨基酸之间显示出普通断点的cpP，以及具有包含用户定义的截短和某些氨基酸重复的新末端的cpP。CiPerGenesis在绿色荧光蛋白（GFP）的盖区G134–H148上进行了测试，结果显示，大多数荧光变体是从Leu141开始，到氨基酸Tyr145，Tyr143，Glu142，Leu141，Lys140和H139终止的那些。某些变体的纯化和生化特性表明存在差异表达，