PURE mRNA display for in vitro selection of single-chain antibodies

Yu Nagumo, Kei Fujiwara, Kenichi Horisawa, Hiroshi Yanagawa, Nobuhide Doi

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

mRNA display is a method to form a covalent linkage between a cell-free synthesized protein (phenotype) and its encoding mRNA (genotype) through puromycin for in vitro selection of proteins. Although a wheat germ cell-free translation system has been previously used in our mRNA display system, a protein synthesis using recombinant elements (PURE) system is a more attractive approach because it contains no endogenous nucleases and proteases and is optimized for folding of antibodies with disulphide bonds. However, when we used the PURE system for mRNA display of single-chain Fv (scFv) antibodies, the formation efficiency of the mRNA-protein conjugates was quite low. To establish an efficient platform for the PURE mRNA display of scFv, we performed affinity selection of a library of scFv antibodies with a C-terminal random sequence and obtained C-terminal sequences that increased the formation of mRNA-protein conjugates. We also identified unexpected common substitution mutations around the start codon of scFv antibodies, which were inferred to destabilize the mRNA secondary structure. This destabilization causes an increase in protein expression and the efficiency of the formation of mRNA-protein conjugates. We believe these improvements should make the PURE mRNA display more efficient for selecting antibodies for diagnostic and therapeutic applications.

Original languageEnglish
Pages (from-to)519-526
Number of pages8
JournalJournal of Biochemistry
Volume159
Issue number5
DOIs
Publication statusPublished - 2016 May 1

Fingerprint

Single-Chain Antibodies
Display devices
Messenger RNA
Proteins
In Vitro Techniques
Puromycin
Initiator Codon
Cell-Free System
Antibodies
Germ Cells
Disulfides
Triticum
Antibody Formation
Peptide Hydrolases
Substitution reactions
Genotype
Cells
Phenotype

Keywords

  • antibody engineering
  • cell-free translation
  • PURE system
  • puromycin
  • ribosome stalling

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

PURE mRNA display for in vitro selection of single-chain antibodies. / Nagumo, Yu; Fujiwara, Kei; Horisawa, Kenichi; Yanagawa, Hiroshi; Doi, Nobuhide.

In: Journal of Biochemistry, Vol. 159, No. 5, 01.05.2016, p. 519-526.

Research output: Contribution to journalArticle

Nagumo, Yu ; Fujiwara, Kei ; Horisawa, Kenichi ; Yanagawa, Hiroshi ; Doi, Nobuhide. / PURE mRNA display for in vitro selection of single-chain antibodies. In: Journal of Biochemistry. 2016 ; Vol. 159, No. 5. pp. 519-526.
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