Purification of VopK Protein by Ni2+-NTA Chromatography

VopK protein was purified by Ni²⁺-nitrilotriacetic acid (Ni²⁺-NTA) chromatography by following a published protocol [12 (link)]. The wild-type gene was cloned into the NdeI-BamHI site of the pET15b vector (Novagen) to generate an N-terminal His₆-VopK fusion protein. The clone was confirmed by sequencing and transformed into E. coli BL21 (DE3). After induction with 0.4mM IPTG (isopropyl1-thio-β-D-galactopyranoside), VopK protein was purified through Qiagen Ni²⁺-NTA column. The purified protein was dialyzed overnight in a solution of buffer A containing 10mM Tris, pH 7.9, 100 mM KCl, 0.1mM EDTA, 0.1mM DTT, 5% glycerol. Protease assay was done essentially as described earlier [13 (link)].

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Bankapalli L.K., Mishra R.C., Singh B, & Raychaudhuri S. (2015). Identification of Critical Amino Acids Conferring Lethality in VopK, a Type III Effector Protein of Vibrio cholerae: Lessons from Yeast Model System. PLoS ONE, 10(10), e0141038.

Publication 2015

Ni2+-NTA column

Assay Buffer Chromatography Clone E coli Edta Galactopyranoside Gene Glycerol Iptg Nitrilotriacetic acid Protease Protein Tris Vector

Corresponding Organization :

Other organizations : Council of Scientific and Industrial Research

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Variable analysis

independent variables

The wild-type gene was cloned into the NdeI-BamHI site of the pET15b vector (Novagen) to generate an N-terminal His6-VopK fusion protein.

dependent variables

Protease assay was done essentially as described earlier [13 (link)].

control variables

The clone was confirmed by sequencing.
The purified protein was dialyzed overnight in a solution of buffer A containing 10mM Tris, pH 7.9, 100 mM KCl, 0.1mM EDTA, 0.1mM DTT, 5% glycerol.

controls

No positive or negative controls were explicitly mentioned.

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