International Journal of Pharmaceutical Investigation, 2020, 10, 2, 117-121.
DOI: 10.5530/ijpi.2020.2.21
Published: June 2020
Type: Original Article
Authors:
Tina Rostinawati
Department of Biology Pharmacy, Faculty of Pharmacy, Padjadjaran University, Sumedang, Jawa Barat, INDONESIA.
Nadia Gitta Paramita
Department of Biology Pharmacy, Faculty of Pharmacy, Padjadjaran University, Sumedang, Jawa Barat, INDONESIA.
Imam Adi Wicaksono
Department of Pharmacology and Clinical Pharmacy, Faculty of Pharmacy, Padjadjaran University, Sumedang, Jawa Barat, INDONESIA.
Sriwidodo S. Sriwidodo
Department of Pharmaceutical and Pharmaceutical Technology, Faculty of Pharmacy, Padjadjaran University, Sumedang, Jawa Barat, INDONESIA.
Muhammad Yusuf
Department of Chemistry, Faculty of Mathematics and Natural Sciences, Padjadjaran University, Sumedang, Jawa Barat, INDONESIA.
Toto Subroto
Department of Chemistry, Faculty of Mathematics and Natural Sciences, Padjadjaran University, Sumedang, Jawa Barat, INDONESIA.
ABSTRACT
Objectives: In patients with breast cancer, Human Epidermal Growth Factor is over expressed until 30%. Monoclonal antibodies was an alternative detection cancer in molecular level. The aim of the experiment was protein recombinant of anti-HER2 scFv was constructed from the gene encoding single chain variable fragment of anti-HER2 antibody wich was fused with Histag and can be expressed in the Eschericia coli BL21(DE3) to be used as a diagnostic protein for breast cancer cells. Methods: The recombinant pJ401express_anti-HER2 scFv fused with histaq was transformed into E. coli BL21 (DE3) and expressed as recombinant anti-HER2 scFv protein with various inducer concentration. Then, those protein was purified with the nickel polyhistidine tag (Ni-NTA) affinity chromatography using imidazole concentration i.e 100 and 150 mM. Finally, the existence of this recombinant protein was determined with anti histaq antibody in western blot assay. Results: Plasmid isolation from E. coli BL21 (DE3) cells revelaed the existence of the recombinant pJ401express_anti-HER2 scFv. The optimum condition for using IPTG as inducer for the intracellular expressed anti-HER2 scFv gene was 1 mM IPTG which was entered into broth medium at the 3.5th hr of growth time of E. coli BL21(DE3). Then, the higher amout of more purified anti-HER2 scFv was obtained using imidazole at 150 mM. The recombinant protein was also bound to anti histaq antibody in western blot assay. Conclusion: the recombinant pJ401express_anti-HER2 scFv was successfully expressed as anti-HER2 scFv protein.
Keywords: Recombinant protein, Fusion protein, Cell breakdown, Purification, Imidazole.