Recombinant single-chain antibodies with various oligopeptide tails for targeted gene delivery

M. Suzuki, A. Takayanagi, N. Shimizu

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

The single-chain antibody (scFv) made by recombinant DNA technology is one of the most useful tools for basic research and clinical applications. To develop a novel targeted gene delivery method, we engineered the scFv gene for the antibody against human epidermal growth factor (EGF) receptor by connecting with DNA sequences for various oligopeptides with negative or positive charges. The resulting recombinant genes encoding artificial scFv with negative or positive tails were expressed in Escherichia coli and yeast Pichia pastris. In E. coli, all the scFv with negatively charged tails were expressed but mainly as an insoluble form, whereas those with positively charged tails were barely expressed. In yeast P. pastris, all the scFv with negatively charged tails were efficiently expressed and secreted into the culture medium. Addition of high salt into the yeast culture increased their secretion. Purification procedure was established for the scFv with the longest negatively charged tail (D4S × 5), yielding 5 mg/l with a purity of over 95%. The scFv-D4S × 5 was designated as a recombinant immunoporter, which was then mixed with plasmid DNA and polyethylenimine (PEI). The resulting DNA/PEI/immunoporter complex (designated recombinant immunogene) exhibited efficient gene delivery to EGF receptor overexpressing A431 tumor cells.

Original languageEnglish
Pages (from-to)781-788
Number of pages8
JournalGene Therapy
Volume10
Issue number9
DOIs
Publication statusPublished - 2003 May

Fingerprint

Single-Chain Antibodies
Oligopeptides
Tail
Polyethyleneimine
Yeasts
Genes
Epidermal Growth Factor Receptor
Escherichia coli
Synthetic Genes
Pichia
Recombinant DNA
DNA
Culture Media
Plasmids
Salts
Technology
Research
Neoplasms

Keywords

  • EGF receptor
  • Immunogene
  • Pichia pastris
  • Recombinant immunoporter
  • scFv antibody
  • Targeting gene therapy

ASJC Scopus subject areas

  • Genetics

Cite this

Recombinant single-chain antibodies with various oligopeptide tails for targeted gene delivery. / Suzuki, M.; Takayanagi, A.; Shimizu, N.

In: Gene Therapy, Vol. 10, No. 9, 05.2003, p. 781-788.

Research output: Contribution to journalArticle

Suzuki, M. ; Takayanagi, A. ; Shimizu, N. / Recombinant single-chain antibodies with various oligopeptide tails for targeted gene delivery. In: Gene Therapy. 2003 ; Vol. 10, No. 9. pp. 781-788.
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