Efficient construction of a large collection of phage-displayed combinatorial peptide libraries
Efficient construction of a large collection of phage-displayed combinatorial peptide libraries
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DOI：
10.2174/1386207054867337
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发表时间：
2005-09-01
影响因子：
1.8
通讯作者：
Kay, BK
中科院分区：
文献类型：
--
作者：
Scholle, MD;Kehoe, JW;Kay, BK
从噬菌体展示的组合肽库中进行选择是鉴定靶蛋白的肽配体的有效策略。用于构建噬菌体展示文库的现有方案利用连接到双链噬菌体DNA中或用单链噬菌粒DNA进行Kunkel诱变。尽管Kunkel方法快速提供了高达10（11）的文库大小，但多达20%的噬菌粒可能是非重组的。通过对当前Kunkel方案的几处修改，我们已经产生了大小高达10（11）个克隆且重组频率接近100%的肽文库。与噬菌粒文库相反，噬菌体文库的产生通过消除对辅助噬菌体的需要而简化了选择实验。我们的方法依赖于在噬菌体M13的基因III的编码区中存在琥珀终止密码子。将含有随机化DNA片段的寡核苷酸退火至噬菌体基因组，使得随机化区域与终止密码子形成异源双链体。然后将寡核苷酸酶促延伸以产生共价闭合的环状DNA，将其电穿孔到大肠杆菌的非抑制菌株中。如果琥珀终止密码子存在于DNA分子中，则蛋白质III不能合成，并且噬菌体不能自我繁殖。该方法可定制用于展示随机或聚焦肽文库。到目前为止，我们已经构建了22个不同的文库，长度范围从8-20个氨基酸，利用完整的或减少的密码子组。
Selections from phage-displayed combinatorial peptide libraries are an effective strategy for identifying peptide ligands to target proteins. Existing protocols for constructing phage-displayed libraries utilize either ligation into double-stranded phage DNA or Kunkel mutagenesis with single-stranded phagemid DNA. Although the Kunkel approach rapidly provides library sizes of up to 10(11), as many as 20% of the phagemids may be non-recombinant. With several modifications to current Kunkel protocols, we have generated peptide libraries with sizes of up to 10(11) clones and recombination frequencies approaching 100%. The production of phage libraries, as opposed to phagemid libraries, simplifies selection experiments by eliminating the need for helper phage. Our approach relies upon the presence of an amber stop codon in the coding region of gene III of bacteriophage M13. Oligonucleotides containing randomized stretches of DNA are annealed to the phage genome such that the randomized region forms a heteroduplex with the stop codon. The oligonucleotide is then enzymatically extended to generate covalently-closed, circular DNA, which is electroporated into a non-suppressor strain of Escherichia coli. If the amber stop codon is present in the DNA molecule, protein III is not synthesized and the phage cannot propagate itself. This method is customizable for the display of either random or focused peptide libraries. To date, we have constructed 22 different libraries ranging from 8-20 amino acids in length, utilizing complete or reduced codon sets.