Recombinant polymer-protein fusion

A promising approach towards efficient and targeted gene delivery

Arash Hatefi, Z. Megeed, H. Ghandehari

Research output: Contribution to journalArticle

40 Citations (Scopus)

Abstract

Background: Synthetic vectors such as polymers have the potential to reduce the safety problems associated with viral vectors; however, their low transfection efficiency limits their clinical utility. To study the critical steps involved in an efficient transgene expression, there is a need for creative approaches that allow a systematic correlation between gene carrier structure and properties necessary for successful gene transfer. Using recombinant techniques a prototype vector comprised of tandem repeating units fused to a targeting moiety was biosynthesized to mediate gene transfer in mammalian cell lines. The carrier was designed to have the structure of (KHKHKHKHKK)6-FGF2 where lysine (K) residues would allow complexation with plasmid DNA, basic fibroblast growth factor (FGF2) to target cells over-expressing FGF2 receptors (FGFR), and histidine (H) residues to facilitate escape from the endosomal compartments. Methods: The gene carrier was biosynthesized in E. coli, purified using a Ni-NTA column, characterized, complexed with pDNA, and the complexes were used to transfect NIH 3T31 T-47D and COS-1 mammalian cell types known to express FGFR. Results: Results demonstrate the successful cloning and expression of the gene carrier with over 95% purity. The molecular weight of the gene carrier was determined by MALDI-TOF to be 27 402. Amino acid content analysis and Western blot confirmed the expression of the gene carrier in E. coli. The vector was able to condense pDNA, induce cell proliferation in NIH 3T3 fibroblasts, and mediate transgene expression in NIH 3T3, T-47D and COS-1 mammalian cell types. Conclusion: Genetic engineering techniques show promise for systematic investigation of structure-activity relationships of non-viral gene delivery vectors.

Original languageEnglish (US)
Pages (from-to)468-476
Number of pages9
JournalJournal of Gene Medicine
Volume8
Issue number4
DOIs
StatePublished - Apr 1 2006
Externally publishedYes

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Recombinant Fusion Proteins
Fibroblast Growth Factor 2
Polymers
Genes
COS Cells
Transgenes
Escherichia coli
Gene Expression
Genetic Techniques
Genetic Engineering
Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
Structure-Activity Relationship
Histidine
Lysine
Transfection
Organism Cloning
Plasmids
Fibroblasts
Molecular Weight
Western Blotting

All Science Journal Classification (ASJC) codes

  • Molecular Medicine
  • Molecular Biology
  • Genetics
  • Drug Discovery
  • Genetics(clinical)

Cite this

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abstract = "Background: Synthetic vectors such as polymers have the potential to reduce the safety problems associated with viral vectors; however, their low transfection efficiency limits their clinical utility. To study the critical steps involved in an efficient transgene expression, there is a need for creative approaches that allow a systematic correlation between gene carrier structure and properties necessary for successful gene transfer. Using recombinant techniques a prototype vector comprised of tandem repeating units fused to a targeting moiety was biosynthesized to mediate gene transfer in mammalian cell lines. The carrier was designed to have the structure of (KHKHKHKHKK)6-FGF2 where lysine (K) residues would allow complexation with plasmid DNA, basic fibroblast growth factor (FGF2) to target cells over-expressing FGF2 receptors (FGFR), and histidine (H) residues to facilitate escape from the endosomal compartments. Methods: The gene carrier was biosynthesized in E. coli, purified using a Ni-NTA column, characterized, complexed with pDNA, and the complexes were used to transfect NIH 3T31 T-47D and COS-1 mammalian cell types known to express FGFR. Results: Results demonstrate the successful cloning and expression of the gene carrier with over 95{\%} purity. The molecular weight of the gene carrier was determined by MALDI-TOF to be 27 402. Amino acid content analysis and Western blot confirmed the expression of the gene carrier in E. coli. The vector was able to condense pDNA, induce cell proliferation in NIH 3T3 fibroblasts, and mediate transgene expression in NIH 3T3, T-47D and COS-1 mammalian cell types. Conclusion: Genetic engineering techniques show promise for systematic investigation of structure-activity relationships of non-viral gene delivery vectors.",
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Recombinant polymer-protein fusion : A promising approach towards efficient and targeted gene delivery. / Hatefi, Arash; Megeed, Z.; Ghandehari, H.

In: Journal of Gene Medicine, Vol. 8, No. 4, 01.04.2006, p. 468-476.

Research output: Contribution to journalArticle

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