Qiu Ji, LaBaer Joshua
Virginia G. Piper Center for Personalized Diagnostics, Biodesign Institute, Arizona State University, Tempe, Arizona, USA.
Methods Enzymol. 2011;500:151-63. doi: 10.1016/B978-0-12-385118-5.00009-8.
Systematic study of proteins requires the availability of thousands of proteins in functional format. However, traditional recombinant protein expression and purification methods have many drawbacks for such study at the proteome level. We have developed an innovative in situ protein expression and capture system, namely NAPPA (nucleic acid programmable protein array), where C-terminal tagged proteins are expressed using an in vitro expression system and efficiently captured/purified by antitag antibodies coprinted at each spot. The NAPPA technology presented in this chapter enable researchers to produce and display fresh proteins just in time in a multiplexed high-throughput fashion and utilize them for various downstream biochemical researches of interest. This platform could revolutionize the field of functional proteomics with it ability to produce thousands of spatially separated proteins in high density with narrow dynamic rand of protein concentrations, reproducibly and functionally.
对蛋白质进行系统研究需要有数千种功能形式的蛋白质。然而,传统的重组蛋白表达和纯化方法在蛋白质组水平的此类研究中有许多缺点。我们开发了一种创新的原位蛋白表达和捕获系统,即核酸可编程蛋白阵列(NAPPA),其中C端标记的蛋白质使用体外表达系统进行表达,并通过在每个点共打印的抗标签抗体进行有效捕获/纯化。本章介绍的NAPPA技术使研究人员能够以多重高通量的方式及时生产和展示新鲜蛋白质,并将其用于各种感兴趣的下游生化研究。该平台能够以高密度、窄动态蛋白质浓度范围、可重复且具有功能地生产数千种空间分离的蛋白质,可能会给功能蛋白质组学领域带来变革。