construction and analysis of high-complexity ribosome display random peptide libraries建设和高复杂性的核糖体的分析显示随机肽库.pdf

Construction and Analysis of High-Complexity Ribosome Display Random Peptide Libraries 2 1 1 1 1 2 Li-Min Yang , Jing-Lin Wang *, Lin Kang , Shan Gao , Yan-hua Liu , Ting-Mao Hu 1 State Key Laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing, China, 2 The Center for Molecular Virology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China Abstract Random peptide libraries displayed on the ribosome are becoming a new tool for the in vitro selection of biologically relevant macromolecules, including epitopes, antagonists, enzymes, and cell-surface receptors. Ribosome display is a cell- free system of coupling individual nascent proteins (phenotypes) to their corresponding mRNA (genotypes) by the formation of stable protein-ribosome-mRNA complexes and permitting the selection of a functional nascent protein by iterative cycles of panning and reverse transcription-polymerase chain reaction (RT-PCR) amplification in vitro. The complexity of the random peptide library is critical for the success of a panning experiment; greater the diversity of sequences within the library, the more likely it is that the library comprises sequences that can bind a given target with specific affinity. Here, we have used the cell-free system Escherichia coli S30 lysate to construct high-complexity random peptide libraries (.1014 independent members) by introducing strategies that are different from the methods described by Mattheakis et al. and Lamla et al. The key step in our method is to produce nanomole (nmol) amounts of DNA elements that are necessary for in vitro transcrip