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- Title
Characterization of Anti-HER2 scFv Gene Expression as Intracellular Protein in Escherichia coli BL21 (DE3).
- Authors
Rostinawati, Tina; Paramita, Nadia Gitta; Wicaksono, Imam Adi; Sriwidodo; Yusuf, Muhammad; Subroto, Toto
- 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.
- Subjects
RECOMBINANT proteins; GENE expression; PROTEIN expression; ESCHERICHIA coli; CHIMERIC proteins
- Publication
International Journal of Pharmaceutical Investigation, 2020, Vol 10, Issue 2, p117
- ISSN
2230-973X
- Publication type
Article
- DOI
10.5530/ijpi.2020.2.21