In vitro selection of fab fragments by mRNA display and gene-linking emulsion PCR

Takeshi Sumida, Hiroshi Yanagawa, Nobuhide Doi

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

In vitro selection by display methods has been an effective tool for engineering recombinant antibodies. mRNA display based on a cell-free translation system has the advantages of larger library sizes and quicker selection procedures compared with cell-based display methods such as phage display. However, mRNA display has been limited to select single-chain polypeptides such as scFvs due to its characteristic of linking a nascent polypeptide with its encoding mRNA on the ribosome. Here we demonstrated a new way of selecting heterodimeric Fab fragments by using mRNA display combined with emulsion PCR. We designed a pair of complementary 5′ UTR sequences that can link the Fab heavy and light chain genes together by overlap-extension PCR in water-in-oil emulsions. We confirmed that two mRNA-displayed polypeptides for heavy and light chain of a model Fab fragment were associated into the active form and that a specific Fab fragment gene was enriched over 100-fold per round of a model affinity selection followed by the gene-linking emulsion PCR. We further performed directed evolution of Fab fragments with higher binding activity from a randomized Fab fragment library.

Original languageEnglish
Article number371379
JournalJournal of Nucleic Acids
Volume2012
DOIs
Publication statusPublished - 2012

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Immunoglobulin Fab Fragments
Emulsions
Genes
Display devices
Polymerase Chain Reaction
Messenger RNA
Peptides
Light
Cell-Free System
5' Untranslated Regions
Bacteriophages
Ribosomes
Libraries
Oils
Telecommunication links
In Vitro Techniques
Water
Antibodies

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

In vitro selection of fab fragments by mRNA display and gene-linking emulsion PCR. / Sumida, Takeshi; Yanagawa, Hiroshi; Doi, Nobuhide.

In: Journal of Nucleic Acids, Vol. 2012, 371379, 2012.

Research output: Contribution to journalArticle

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