title: Co-expression of functional human Heme Oxygenase 1, Ecto-5'-Nucleotidase and ecto-nucleoside triphosphate diphosphohydrolase-1 by "self-cleaving" 2A peptide system.
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.
authors.affiliation: Department of Surgery and Translational Medicine, University of Milano-Bicocca, Monza, Italy; Department of Biochemistry, Medical University of Gdansk, Gdansk, Poland.
authors.forename: Marco
authors.initials: M
authors.lastname: De Giorgi
authors.affiliation: Department of Surgery and Translational Medicine, University of Milano-Bicocca, Monza, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Cinti
authors.affiliation: Department of Biochemistry, Medical University of Gdansk, Gdansk, Poland.
authors.forename: Iwona
authors.initials: I
authors.lastname: Pelikant-Malecka
authors.affiliation: Department of Surgery and Translational Medicine, University of Milano-Bicocca, Monza, Italy.
authors.forename: Elisa
authors.initials: E
authors.lastname: Chisci
authors.affiliation: Department of Surgery and Translational Medicine, University of Milano-Bicocca, Monza, Italy.
authors.forename: Marialuisa
authors.initials: M
authors.lastname: Lavitrano
authors.affiliation: Department of Surgery and Translational Medicine, University of Milano-Bicocca, Monza, Italy.
authors.forename: Roberto
authors.initials: R
authors.lastname: Giovannoni
authors.affiliation: Department of Surgery and Translational Medicine, University of Milano-Bicocca, Monza, Italy; Department of Biochemistry, Medical University of Gdansk, Gdansk, Poland. Electronic address: rt.smolenski@gumed.edu.pl.
authors.forename: Ryszard T
authors.initials: RT
authors.lastname: Smolenski
mesh_terms: D015720:5'-Nucleotidase / Q000235:genetics* / Q000378:metabolism; D015703:Antigens, CD / Q000235:genetics* / Q000378:metabolism; D001081:Apyrase / Q000235:genetics* / Q000378:metabolism; D045683:Furin / Q000378:metabolism; D058851:GPI-Linked Proteins / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D051547:Heme Oxygenase-1 / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D010957:Plasmids / Q000235:genetics; D014183:Transplantation, Heterologous
chemical_list: D015703:Antigens, CD; D058851:GPI-Linked Proteins; C496541:HMOX1 protein, human; D051547:Heme Oxygenase-1; D015720:5'-Nucleotidase; C548241:NT5E protein, human; D045683:Furin; D001081:Apyrase; C067869:CD39 antigen
keywords: 2A peptide; Ecto 5′ nucleotidase; Ecto nucleoside triphosphate diphosphohydrolase 1; Heme oxygenase 1; Multicistronic vector; Xenotransplantation