Co-expression of functional human Heme Oxygenase 1, Ecto-5'-Nucleotidase and ecto-nucleoside triphosphate diphosphohydrolase-1 by "self-cleaving" 2A peptide system

Plasmid. 2015 May;79:22-9. doi: 10.1016/j.plasmid.2015.03.004. Epub 2015 Mar 14.

Abstract

We developed an F2A-based multicistronic system to evaluate functional effects of co-expression of three proteins important for xenotransplantation: heme oxygenase 1 (HO1), ecto-5'-nucleotidase (E5NT) and ecto-nucleoside triphosphate diphosphohydrolase-1 (ENTPD1). The tricistronic p2A plasmid that we constructed was able to efficiently drive concurrent expression of HO1, E5NT and ENTPD1 in HEK293T cells. All three overexpressed proteins possessed relevant enzymatic activities, while addition of furin site interfered with protein expression and activity. We conclude that our tricistronic p2A construct is effective and optimal to test the combined protective effects of HO1, E5NT and ENTPD1 against xeno-rejection mechanisms.

Keywords: 2A peptide; Ecto 5′ nucleotidase; Ecto nucleoside triphosphate diphosphohydrolase 1; Heme oxygenase 1; Multicistronic vector; Xenotransplantation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 5'-Nucleotidase / genetics*
  • 5'-Nucleotidase / metabolism
  • Antigens, CD / genetics*
  • Antigens, CD / metabolism
  • Apyrase / genetics*
  • Apyrase / metabolism
  • Furin / metabolism
  • GPI-Linked Proteins / genetics
  • GPI-Linked Proteins / metabolism
  • Gene Expression Regulation*
  • HEK293 Cells
  • Heme Oxygenase-1 / genetics*
  • Heme Oxygenase-1 / metabolism
  • Humans
  • Plasmids / genetics
  • Transplantation, Heterologous

Substances

  • Antigens, CD
  • GPI-Linked Proteins
  • HMOX1 protein, human
  • Heme Oxygenase-1
  • 5'-Nucleotidase
  • NT5E protein, human
  • Furin
  • Apyrase
  • CD39 antigen