Characterization of Anti-HER2 scFv Gene Expression as Intracellular Protein in Escherichia coli BL21 (DE3)

  • 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.
Keywords: Recombinant protein, Fusion protein, Cell breakdown, Purification, Imidazole

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.

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Figure 1: Map of Construction of pJ401express_anti-HER2 scFv shynthetic gene
Published
2020-06-08
How to Cite
1.
Rostinawati T, Paramita NG, Wicaksono IA, S. Sriwidodo S, Yusuf M, Subroto T. Characterization of Anti-HER2 scFv Gene Expression as Intracellular Protein in Escherichia coli BL21 (DE3). ijpi [Internet]. 8Jun.2020 [cited 7Jul.2020];10(2):117-21. Available from: http://www.jpionline.org/index.php/ijpi/article/view/460