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Cell-Free Protein Production

Volume 607 of the series Methods in Molecular Biology pp 219-225

Date:

Ribosome Display with the PURE Technology

  • Takuya UedaAffiliated withThe Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo
  • , Takashi KanamoriAffiliated withThe Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo
  • , Hiroyuki OhashiAffiliated withThe Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo

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Abstract

The ribosome display utilizes the formation of the mRNA–ribosome–polypeptide ternary complex in the cell-free protein synthesis system as the linking of the genotype (mRNA) to the phenotype (polypeptide). However, the presence of intrinsic components such as nucleases in the cell-extract based cell-free protein synthesis systems inevitably reduces the stability of the ternary complex, which would prevent attainment of reliable results. We have developed an efficient and highly controllable ribosome display system using the Protein synthesis Using Recombinant Elements (PURE) system. The mRNA–ribosome–polypeptide ternary complex is highly stable in the PURE system and then the selected mRNA can be easily recovered, because activities of nucleases and other inhibitory factors are very low in the PURE system. Furthermore, omission of the release factors within the original PURE system can aid stalling of the ribosome at the termination codon to form the mRNA–ribosome–polypeptide ternary complex. We believe that these advantages assure the usability of the modified PURE system for ribosome display.

Key words

PURE system Ribosome display Cell-free protein synthesis system In vitro selection Single chain Fv Protein engineering