CONSTRUCTION OF IN VITRO EVOLUTION SYSTEM OF PROTEINS

已经开发了一种新的用于产生蛋白质文库的体外方法，该方法使用单个DNA分子的聚合酶链式反应（PCR）扩增，随后进行体外偶联转录/翻译。将编码维多利亚水母（Aequorea victoria）的绿色荧光蛋白（GFP）的DNA模板广泛稀释至每管一个分子，然后通过使用巢式引物的总共80个循环的PCR扩增。然后通过直接测序片段的一部分来估计扩增的DNA片段中的确切起源数，在该片段处，通过PCR用含有三个随机碱基的引物标记单个模板分子。由于91个独立扩增的序列在统计学上是多样化的，每个扩增片段可能来自单个DNA分子。此外，扩增的片段用作使用T7 RNA聚合酶和大肠杆菌S30提取物的体外偶联转录/翻译的模板。这些结果表明，通过PCR扩增从各种DNA池稀释的单个DNA分子获得的文库在高通量产生蛋白质文库中是潜在有用的。

A novel in vitro method for the generation of a protein library has been developed using the polymerase chain reaction (PCR) amplification of a single DNA molecule followed by in vitro coupled transcription/translation. DNA template encoding green fluorescent protein (GFP) of a jellyfish Aequorea victoria was extensively diluted to one molecule per tube, and then amplified by a total of 80 cycles of PCR with nested primers. The exact number of origins in the amplified DNA fragment was then estimated by directly sequencing a part of the fragment, at which an individual template molecule was marked by PCR with a primer containing three randomized bases. Since the sequehces obtained in 91 independent amplifications were diversified statistically, each amplified fragment was likely originated from a single DNA molecule. In addition, the amplified fragments served as a template for in vitro coupled transcription/translation using T7 RNA polymerase and E.ccli S30 extract. These results suggest that the library obtained by the PCR amplification of a single DNA molecule diluted from a variety of DNA pools is potentially useful in high-throughput generation of protein libraries.