Construction and Analysis of High-Complexity Ribosome Display Random Peptide Libraries

Li-Min Yang,Yan-Hua Liu,Jing-Lin Wang,Lin Kang,Shan Gao,Ting-Mao Hu,Thomas Zwaka

doi:10.1371/journal.pone.0002092

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 transcription/translation by using PCR but not plasmid DNA. Library design strategies and protocols that facilitate rapid identification are also presented.

Highlights

Since the mid-1980s, a number of different methods have been developed to screen peptide or protein libraries for specific binders
A majority of these methods, such as phage display [1], cell surface display [2,3], plasmid display [4], and the yeast twohybrid system [5], are so-called in vivo systems because living cells are involved in these processes of library generation or screening
Ribosome display is an in vitro technology for the simultaneous selection and evolution of proteins from diverse libraries. This technology relies on non-covalent ternary polypeptide-ribosome-mRNA complexes, which ensures the coupling of genotypes and phenotypes

Summary

Introduction

Since the mid-1980s, a number of different methods have been developed to screen peptide or protein libraries for specific binders. We comprehensively report the construction of highcomplexity random peptide libraries with 1.261014 independent members based on the scaffold of amino acids T20–V109 of protein D (pD), a structured part of the capsid protein from phage Lambda, by using in vitro ribosome display technology. (pD) (a structured part of the capsid protein from phage Lambda, possessing 12 randomized amino acid residues at its N terminus) was generated by introducing the elements necessary for its efficient in vitro transcription/translation to a DNA fragment encoding 12 NNK codons through DNA ligation.

Results

Conclusion