Phage display peptide libraries: deviations from randomness and correctives.
Phage display peptide libraries: deviations from randomness and correctives.
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DOI：
10.1093/nar/gky077
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发表时间：
2018-05-18
影响因子：
14.9
通讯作者：
Gershoni JM
中科院分区：
文献类型：
--
作者：
Ryvkin A;Ashkenazy H;Weiss-Ottolenghi Y;Piller C;Pupko T;Gershoni JM
表达肽的噬菌体展示文库广泛用于抗体的检测。然后分析亲和选择的肽以发现表位模拟物，或进行表位预测的计算算法。这些应用的一个关键假设是初始天然肽库中氨基酸的随机表示。在之前的一项研究中，我们实施了下一代测序来评估初始文库，发现 UAG 密码子过度表达以及高 G 亚磷酰胺丰度与随机性存在严重偏差，导致氨基酸分布偏差。在本研究中，我们证明 UAG 过度表达可归因于重组蛋白 8 亚基组装时噬菌体承受的负担。通过使用含有supE44的细菌构建文库来纠正这一问题，该细菌抑制UAG驱动的流产终止。我们还证明，G 的过量源于变体合成效率，并且可以使用通过质谱校准的补偿性寡核苷酸混合物进行校正。实施这些校正的文库构建可显着改进文库，显示氨基酸的随机分布，从而确保生物淘选中获得的富集肽代表真正的选择事件，这是噬菌体展示应用的基本假设。
Peptide-expressing phage display libraries are widely used for the interrogation of antibodies. Affinity selected peptides are then analyzed to discover epitope mimetics, or are subjected to computational algorithms for epitope prediction. A critical assumption for these applications is the random representation of amino acids in the initial naïve peptide library. In a previous study, we implemented next generation sequencing to evaluate a naïve library and discovered severe deviations from randomness in UAG codon over-representation as well as in high G phosphoramidite abundance causing amino acid distribution biases. In this study, we demonstrate that the UAG over-representation can be attributed to the burden imposed on the phage upon the assembly of the recombinant Protein 8 subunits. This was corrected by constructing the libraries using supE44-containing bacteria which suppress the UAG driven abortive termination. We also demonstrate that the overabundance of G stems from variant synthesis-efficiency and can be corrected using compensating oligonucleotide-mixtures calibrated by mass spectroscopy. Construction of libraries implementing these correctives results in markedly improved libraries that display random distribution of amino acids, thus ensuring that enriched peptides obtained in biopanning represent a genuine selection event, a fundamental assumption for phage display applications.
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影响因子：
3.7
作者：
Dias-Neto E;Nunes DN;Giordano RJ;Sun J;Botz GH;Yang K;Setubal JC;Pasqualini R;Arap W
通讯作者：
Arap W
影响因子：
7.3
作者：
Deschaght, Pieter;Vintem, Ana Paula;Dombrecht, Bruno
通讯作者：
Dombrecht, Bruno
影响因子：
3.7
作者：
Frietze KM;Pascale JM;Moreno B;Chackerian B;Peabody DS
通讯作者：
Peabody DS
影响因子：
3.7
作者：
Brinton LT;Bauknight DK;Dasa SS;Kelly KA
通讯作者：
Kelly KA
DOI：
10.1073/pnas.1517813113
发表时间：
2016-03-29
影响因子：
11.1
作者：
Boyer, Sebastien;Biswas, Dipanwita;Rivoire, Olivier
通讯作者：
Rivoire, Olivier