Studying of Membrane Localization of Recombinant Potassium Channels in                   E.coli

Nekrasova, Oksana V.; Tagway, A.; Ignatova, Anastasia A.; Feofanov, Аlexey V.; Kirpichnikov, Mikhaǐl P.

doi:10.32607/20758251-2009-1-1-91-95

Cited by 8 publications

(1 citation statement)

References 25 publications

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“…9 Some literature shows the advantages of extracellular protein growth to move from the cell growth phase to the phase of protein production by adding the IPTG inducer. 21,23,24 Purification result with Ni-NTA affinity chromatography showed the presence of non-target proteins that were eluted from the column. This is due to some cellular proteins contain two or more adjacent histidine residues.…”

Section: Discussionmentioning

confidence: 99%

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

Rostinawati

Paramita

Wicaksono

et al. 2020

IJPI

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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 3.5 th 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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Section: Discussionmentioning

confidence: 99%