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

在此，我们开发了一种新的蛋白质文库系统，称为SIMPLEX：单分子PCR连接体外表达，结合PCR扩增的单个DNA分子稀释在井和无细胞蛋白质合成。使用单引物扩增和热启动DNA聚合酶实现单步单分子PCR。通过SIMPLEX获得的蛋白库具有高度的一致性。均匀性实际上比传统的体内系统高得多。此外，井中的多分子PCR已被证明是可能的。这些结果表明SIMPLEX可以为蛋白质文库的构建和筛选提供高通量的方法。此外，还可以在浓度为10,000/cm^2的微室阵列上合成蛋白质，这将为高浓缩蛋白文库的构建奠定基础。此外，体外组合诱变也被开发出来，以证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特性。

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.