Lin Zengxi, Wang Shenglan, Yang Xiushan, Yang Keqian
Capital Normal University, Beijing 100037, China.
Sheng Wu Gong Cheng Xue Bao. 2008 Nov;24(11):1924-30.
A library with potential to produce six amino acids cyclic peptides was prepared using pET-28a as the starting plasmid. pVmut was used to amplify the Int(C)-dnaB-N-Int(N) fragment that was inserted into pET28a to give pEV. On pEV, DnaB split intein was expressed under the strong T7 promoter. Analyses of Escherichia coli transformed with pEV showed that DnaB split intein was produced in large quantity and the fusion protein self-spliced efficiently to produce cyclized DnaB-N. A synthesized 115 bp fragment mixture encoding 5 random amino acids was inserted into pEV to generate pEV-IS. The ligation mixture was transformed into E. coli. A library of 10(3) clones was obtained, 20 randomly picked clones were sequenced. All of them contain different sequences. Nine clones were chosen for further analysis. Split-intein-ISs were expressed in large quantity, and 90% of them self-spliced under 16 degrees C in 20 hours. After induction at 30 degrees C for 3 hours, the expressed DnaB split intein was purified using His-column, and then a molecular weight of target cyclic peptide was detected by MALDI-TOF-MS.
以pET-28a为起始质粒构建了一个有潜力产生六种氨基酸环肽的文库。使用pVmut扩增Int(C)-dnaB-N-Int(N)片段，将其插入pET28a得到pEV。在pEV上，DnaB分裂内含肽在强T7启动子下表达。对用pEV转化的大肠杆菌进行分析表明，DnaB分裂内含肽大量产生，融合蛋白高效自剪接产生环化的DnaB-N。将编码5个随机氨基酸的合成115 bp片段混合物插入pEV以产生pEV-IS。连接混合物转化到大肠杆菌中。获得了一个包含10³个克隆的文库，随机挑选20个克隆进行测序。它们都包含不同的序列。选择9个克隆进行进一步分析。分裂内含肽-IS大量表达，其中90%在16℃下20小时内自剪接。在30℃诱导3小时后，用His柱纯化表达的DnaB分裂内含肽，然后通过基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）检测目标环肽的分子量。
