Single step generation of protein arrays from DNA by cell-free expression and in situ immobilisation (PISA method)
Single step generation of protein arrays from DNA by cell-free expression and in situ immobilisation (PISA method)
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DOI：
10.1093/nar/29.15.e73
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发表时间：
2001-08-01
影响因子：
14.9
通讯作者：
Taussig, MJ
中科院分区：
文献类型：
--
作者：
He, MY;Taussig, MJ
我们描述了一种生产蛋白质阵列的格式，称为“蛋白质原位阵列”（比萨）。比萨通过无细胞蛋白表达和在表面的原位固定化直接从PCR产生的DNA片段在一个步骤中快速产生。用于表达的模板是编码单个蛋白质或结构域的DNA，其通过使用根据DNA数据库中的信息设计的引物的PCR产生。偶联的转录和翻译在标记蛋白质一合成就粘附的表面上进行。由于通过无细胞合成产生的蛋白质通常是可溶性和功能性的，因此该方法可以克服与重组蛋白的细菌表达相关的不溶性或降解问题。此外，使用PCR产生的DNA能够仅基于基因组信息快速产生蛋白质或结构域，并且在克隆材料不可用的情况下将特别有用。在这里，我们表明，人类单链抗体片段（三个结构域，V-H/K形式）和酶（荧光素酶）可以功能性排列的比萨方法。
We describe a format for production of protein arrays termed 'protein in situ array' (PISA). A PISA is rapidly generated in one step directly from PCR-generated DNA fragments by cell-free protein expression and in situ immobilisation at a surface. The template for expression is DNA encoding individual proteins or domains, which Is produced by PCR using primers designed from information in DNA databases. Coupled transcription and translation is carried out on a surface to which the tagged protein adheres as soon as it is synthesised. Because proteins generated by cell-free synthesis are usually soluble and functional, this method can overcome problems of insolubility or degradation associated with bacterial expression of recombinant proteins. Moreover, the use of PCR-generated DNA enables rapid production of proteins or domains based on genome Information alone and will be particularly useful where cloned material is not available. Here we show that human single-chain antibody fragments (three domain, V-H/K form) and an enzyme (luciferase) can be functionally arrayed by the PISA method.