Prognosys Biosciences Inc., La Jolla, California, United States of America.
PLoS One. 2012;7(6):e37441. doi: 10.1371/journal.pone.0037441. Epub 2012 Jun 12.
We report a scalable and cost-effective technology for generating and screening high-complexity customizable peptide sets. The peptides are made as peptide-cDNA fusions by in vitro transcription/translation from pools of DNA templates generated by microarray-based synthesis. This approach enables large custom sets of peptides to be designed in silico, manufactured cost-effectively in parallel, and assayed efficiently in a multiplexed fashion. The utility of our peptide-cDNA fusion pools was demonstrated in two activity-based assays designed to discover protease and kinase substrates. In the protease assay, cleaved peptide substrates were separated from uncleaved and identified by digital sequencing of their cognate cDNAs. We screened the 3,011 amino acid HCV proteome for susceptibility to cleavage by the HCV NS3/4A protease and identified all 3 known trans cleavage sites with high specificity. In the kinase assay, peptide substrates phosphorylated by tyrosine kinases were captured and identified by sequencing of their cDNAs. We screened a pool of 3,243 peptides against Abl kinase and showed that phosphorylation events detected were specific and consistent with the known substrate preferences of Abl kinase. Our approach is scalable and adaptable to other protein-based assays.
我们报告了一种可扩展且具有成本效益的技术，用于生成和筛选高复杂度的定制肽组。这些肽通过体外转录/翻译从基于微阵列合成的 DNA 模板库中制成肽-cDNA 融合物。这种方法可以在计算机上设计大型定制肽组，以有效的并行方式进行成本效益制造，并以多路复用方式进行高效检测。我们的肽-cDNA 融合池在两种基于活性的测定中得到了验证，这些测定旨在发现蛋白酶和激酶底物。在蛋白酶测定中，通过其同源 cDNA 的数字测序，将切割的肽底物与未切割的底物分离并鉴定。我们对 HCV 蛋白酶体中的 3011 个氨基酸进行了易感性筛选，以确定 HCV NS3/4A 蛋白酶的切割，并高度特异性地鉴定了所有 3 个已知的跨切割位点。在激酶测定中，通过其 cDNA 的测序来捕获和鉴定由酪氨酸激酶磷酸化的肽底物。我们针对 Abl 激酶筛选了 3243 个肽的池，并表明检测到的磷酸化事件是特异性的，与 Abl 激酶的已知底物偏好一致。我们的方法具有可扩展性，并且可以适应其他基于蛋白质的测定。
