Construction of highly condensed protein/peptide library on micro-reactor array

在这里，我们开发了一种新的蛋白质文库系统，称为SIMPLEX：单分子PCR连接的体外表达，结合了在井中稀释的单个DNA分子的PCR扩增和无细胞蛋白质合成。利用单引物扩增和热启动DNA聚合酶，实现了单步单分子聚合酶链式反应。单纯形法获得的蛋白质文库具有高度的均一性。这种均匀性实际上比传统的体内系统获得的要高得多。此外，在井中进行多分子聚合酶链式反应也已被证明是可能的。这些结果表明，单纯形法可以为蛋白质文库的构建和筛选提供一种高通量的方法。此外，蛋白质可以在浓度为10,000/cm2的微腔阵列上合成，这将导致构建高度浓缩的蛋白质文库；此外，体外组合诱变还表明，根据蛋白质的三维结构选择特定区域的组合突变可以有效地调节或改善蛋白质的某些性质。

Herein we have developed a novel protein library system termed SIMPLEX : single-molecule PCR linked in vitro expression combining PCR amplification of single DNA molecule diluted in a well and cell-free protein synthesis. Use of single primer amplification and hot-startable DNA polymerase enabled a single step single-molecule PCR. The obtained protein library by SIMPLEX has been demonstrated to be highly uniform. The uniformity was actually much higher than that obtained by a conventional in vivo system. Also multi-molecule PCR in a well has been shown to be possible. These results have demonstrated that SIMPLEX can provide a high-throughput method for the construction and screening of protein library. Moreover, protein could be synthesized on a micro-chamber array with the concentration of 10,000/cm^2, which will lead the construction of highly condensed protein library.In addition, in vitro combinatorial mutagenesis was also developed to demonstrate that combinatorial mutation in a specific region of protein selected based on its 3D structure is effective to modulate or improve some properties of proteins.