Yáñez Jorge, Argüello Martha, Osuna Joel, Soberón Xavier, Gaytán Paul
Instituto de Biotecnología/UNAM, Ap. Postal 510-3 Cuernavaca, Morelos 62250, México.
Nucleic Acids Res. 2004 Nov 10;32(20):e158. doi: 10.1093/nar/gnh156.
Twenty Fmoc-protected trinucleotide phosphoramidites representing a complete set of codons for the natural amino acids were chemically synthesized for the first time. A pool of these reagents was incorporated into oligonucleotides at substoichiometric levels to generate two libraries of variants that randomly carry either few or many codon replacements on a region encoding nine amino acids of the bacterial enzyme TEM-1 beta-lactamase. Assembly of the libraries was performed in a completely automated mode through a simple modification of ordinary protocols. This technology eliminates codon redundancy, stop codons and enables complete exploration of sequence space for single, double and triple mutations throughout a protein region spanning several residues. Sequence analysis of many non-selected clones revealed a good incorporation of the trinucleotides, producing combinations of mutations quite different from those obtained using conventional degenerate oligonucleotides. Ceftazidime-selection experiments yielded several never before reported variants containing novel amino acid combinations in the beta-lactamase omega loop region.
首次化学合成了代表天然氨基酸完整密码子集的20种Fmoc保护的三核苷酸亚磷酰胺。将这些试剂库以亚化学计量水平掺入寡核苷酸中，以生成两个变体文库，这些文库在编码细菌酶TEM-1β-内酰胺酶九个氨基酸的区域上随机携带少量或多个密码子替换。通过对常规方案进行简单修改，以完全自动化的模式进行文库组装。该技术消除了密码子冗余、终止密码子，并能够在跨越多个残基的蛋白质区域中对单、双和三突变的序列空间进行全面探索。对许多未选择克隆的序列分析表明，三核苷酸的掺入效果良好，产生的突变组合与使用传统简并寡核苷酸获得的突变组合有很大不同。头孢他啶筛选实验产生了几个以前从未报道过的变体，这些变体在β-内酰胺酶ω环区域含有新的氨基酸组合。
