Next-generation phage display: integrating and comparing available molecular tools to enable cost-effective high-throughput analysis.
Next-generation phage display: integrating and comparing available molecular tools to enable cost-effective high-throughput analysis.
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DOI：
10.1371/journal.pone.0008338
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发表时间：
2009-12-17
期刊：
影响因子：
3.7
通讯作者：
Arap W
中科院分区：
文献类型：
--
作者：
Dias-Neto E;Nunes DN;Giordano RJ;Sun J;Botz GH;Yang K;Setubal JC;Pasqualini R;Arap W
在过去的20年里，组合噬菌体展示被用于鉴定蛋白质-配体和蛋白质-蛋白质相互作用，揭示了相关的分子识别事件。组合噬菌体展示文库选择的限速步骤是(I)转导单元的计数和(Ii)编码展示配体的测序。在这里，我们采用了新兴的基因组技术，以最大限度地减少此类挑战。我们通过应用串联实时PCR进行快速定量来实现无菌噬菌体展示文库筛选，并增加了用于大规模配基分析的噬菌体DNA下一代测序，从而提高了效率，报告了一套完全集成的高通量定量和分析工具。该方法的劳动密集度要低得多，并允许严格的量化；对于医疗应用，包括对患者的选择，它也代表着在更长的时间范围内对来自患者的活检或尸检的数十万目标颗粒进行定量分布分析和配体识别的进步。与目前的方法相比，其他优点包括更高的灵敏度、更小的变异性、更强的线性度、可扩展性和准确性，而成本要低得多。QPhage+焦磷酸测序得到的序列与传统的Sanger测序转导单元(TU)得到的数据集相似，没有因GC含量、密码子使用和氨基酸或多肽频率而产生的偏差。这些工具可以使噬菌体展示选择和配基分析的速度提高1,000倍，并将生成106个配基序列的成本∼降低250倍。我们的分析表明，虽然这种方法与传统的菌落计数方法相关，但它也能够进行更大的采样，从而允许对噬菌体浓缩进行更快、更便宜、更准确和一致的分析。总体而言，qPhage加焦糖测序优于TU计数加Sanger测序，被认为是在体外、细胞和体内广泛应用的噬菌体展示方法的选择。
Combinatorial phage display has been used in the last 20 years in the identification of protein-ligands and protein-protein interactions, uncovering relevant molecular recognition events. Rate-limiting steps of combinatorial phage display library selection are (i) the counting of transducing units and (ii) the sequencing of the encoded displayed ligands. Here, we adapted emerging genomic technologies to minimize such challenges. We gained efficiency by applying in tandem real-time PCR for rapid quantification to enable bacteria-free phage display library screening, and added phage DNA next-generation sequencing for large-scale ligand analysis, reporting a fully integrated set of high-throughput quantitative and analytical tools. The approach is far less labor-intensive and allows rigorous quantification; for medical applications, including selections in patients, it also represents an advance for quantitative distribution analysis and ligand identification of hundreds of thousands of targeted particles from patient-derived biopsy or autopsy in a longer timeframe post library administration. Additional advantages over current methods include increased sensitivity, less variability, enhanced linearity, scalability, and accuracy at much lower cost. Sequences obtained by qPhage plus pyrosequencing were similar to a dataset produced from conventional Sanger-sequenced transducing-units (TU), with no biases due to GC content, codon usage, and amino acid or peptide frequency. These tools allow phage display selection and ligand analysis at >1,000-fold faster rate, and reduce costs ∼250-fold for generating 106 ligand sequences. Our analyses demonstrates that whereas this approach correlates with the traditional colony-counting, it is also capable of a much larger sampling, allowing a faster, less expensive, more accurate and consistent analysis of phage enrichment. Overall, qPhage plus pyrosequencing is superior to TU-counting plus Sanger sequencing and is proposed as the method of choice over a broad range of phage display applications in vitro, in cells, and in vivo.
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影响因子：
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