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

M. Suzuki, A. Takayanagi, N. Shimizu

研究成果: Article査読

7 被引用数 (Scopus)

抄録

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.

本文言語English
ページ(範囲)781-788
ページ数8
ジャーナルGene Therapy
10
9
DOI
出版ステータスPublished - 2003 5

ASJC Scopus subject areas

  • 分子医療
  • 分子生物学
  • 遺伝学

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