A new vector for controllable expression of an anti-HER2/neu mini-antibody-barnase fusion protein in HEK 293T cells

Elena M. Glinka, Eweline F. Edelweiss, Alexander M. Sapozhnikov, Sergey M. Deyev

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

Tumor-targeted vectors with controllable expression of therapeutic genes and specific antitumor antibodies are promising tools for the reduction of malignant tumors. Here we describe a new plasmid for the eukaryotic expression of an anti-HER2/neu mini-antibody-barnase fusion protein (4D5 scFv-barnase-His5) with an NH2-terminal leader peptide. The 4D5 scFv-barnase-His5 gene was placed downstream of the tetracycline responsive-element minimal promoter in the vector using the Tet-Off gene-expression system. The Bacillus amyloliquefaciens ribonuclease barnase is toxic for the host cells. To overcome this problem, barstar gene under its own minimal cytomegalovirus promoter was used in designed vector. Barstar inhibits the background level of barnase in the cells in the presence of tetracycline in culture medium. The HEK 293T cells were transfected with the designed vector, and the 4D5 scFv-barnase-His5 fusion protein was identified by anti-barnase antibodies in cell culture medium and after purification from cell lysates using metal-affinity chromatography. The overexpression of the anti-HER2/neu mini-antibody-barnase fusion protein decreased the intensity of fluorescence of HEK 293T cells co-transfected with the generated plasmid and a plasmid containing the gene of enhanced green fluorescent protein (pEGFP-N1), in comparison with the intensity of fluorescence of HEK 293T cells transfected with pEGFP-N1, in the absence of tetracycline in the medium. The effect of the 4D5 scFv-barnase-His5 on EGFP fluorescence indicates that the introduced barnase functions as a ribonuclease inside the cells. The anti-HER2/neu mini-antibody could be used to deliver barnase to HER2/neu-positive cells and provide its penetration into the target cells, as HER2/neu is a ligand-internalizing receptor. This expression vector has potential applications to both gene and antibody therapies of cancer.

Original languageEnglish
Pages (from-to)97-103
Number of pages7
JournalGene
Volume366
Issue number1
DOIs
Publication statusPublished - 17 Jan 2006
Externally publishedYes

Fingerprint

HEK293 Cells
Antibodies
Proteins
Tetracycline
Plasmids
Fluorescence
Culture Media
Bacillus amyloliquefaciens ribonuclease
Genes
Gene Expression
Neoplasms
Poisons
Ribonucleases
Protein Sorting Signals
Cytomegalovirus
Affinity Chromatography
Genetic Therapy
Anti-Idiotypic Antibodies
Cell Culture Techniques
Metals

Keywords

  • Barstar
  • Fusion protein
  • Leader peptide
  • scFv 4D5
  • Transactivator

ASJC Scopus subject areas

  • Medicine(all)
  • Genetics

Cite this

A new vector for controllable expression of an anti-HER2/neu mini-antibody-barnase fusion protein in HEK 293T cells. / Glinka, Elena M.; Edelweiss, Eweline F.; Sapozhnikov, Alexander M.; Deyev, Sergey M.

In: Gene, Vol. 366, No. 1, 17.01.2006, p. 97-103.

Research output: Contribution to journalArticle

Glinka, Elena M. ; Edelweiss, Eweline F. ; Sapozhnikov, Alexander M. ; Deyev, Sergey M. / A new vector for controllable expression of an anti-HER2/neu mini-antibody-barnase fusion protein in HEK 293T cells. In: Gene. 2006 ; Vol. 366, No. 1. pp. 97-103.
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