He Mingyue, Stoevesandt Oda
The Babraham Institute, Cambridge, UK.
Methods Mol Biol. 2010;615:345-56. doi: 10.1007/978-1-60761-535-4_24.
Polypeptide and protein arrays enable high-throughput screening capabilities for studying molecular interactions and profiling of biomarkers, and provide a powerful functional screening tool for peptidomics. To overcome the limitations of conventional arraying methods, we have exploited cell-free systems for generating arrays of polypeptides by direct on-chip biosynthesis from DNA templates. Here we describe two protocols: (i) Protein In Situ Array (PISA), which allows the generation of polypeptide arrays in a single reaction by spotting cell-free lysate together with PCR DNA on a glass surface pre-coated with a capturing reagent, and (ii) DNA Array to Protein Array (DAPA), which is capable of producing multiple copies of a polypeptide array from a single DNA array template. The main advantage of these methods is in using an in vitro coupled transcription and translation system which circumvents the need to synthesise and purify individual polypeptides. Our methods allow making polypeptide arrays using amplified linear DNA fragments.
多肽和蛋白质阵列能够实现高通量筛选,用于研究分子相互作用和生物标志物的分析,为肽组学提供了强大的功能筛选工具。为了克服传统阵列方法的局限性,我们利用无细胞系统通过从DNA模板直接在芯片上进行生物合成来生成多肽阵列。在此,我们描述两种方法:(i)蛋白质原位阵列(PISA),它通过将无细胞裂解物与PCR DNA一起点样在预先涂有捕获试剂的玻璃表面上,在单个反应中生成多肽阵列;(ii)DNA阵列到蛋白质阵列(DAPA),它能够从单个DNA阵列模板产生多肽阵列的多个副本。这些方法的主要优点在于使用体外耦合转录和翻译系统,从而避免了合成和纯化单个多肽的需要。我们的方法允许使用扩增的线性DNA片段制作多肽阵列。