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Figure 4 | Mobile DNA

Figure 4

From: Soluble expression, purification and characterization of the full length IS2 Transposase

Figure 4

12% SDS-PAGE analysis of proteins prepared under native conditions. (A) Analysis of fluorometrically determined peak fractions from Ni-NTA gravity flow affinity chromatography purification of the 6xHis-tagged OrfAB-GFP. Lanes: 1. Prestained Protein Molecular Weight markers (New England Biolabs). 2-4. Partial purification of the 74 kDa His-tagged OrfAB-GFP fusion protein (upper arrow) from cells with the pLL2522 plasmid. The lower arrow identifies the 17.5 kDa OrfA protein generated by programmed -1 translational frameshifting. These lanes represent peak fractions (determined fluorometrically) which were run out prior to pooling. (B) Analysis of the pooled fractions in part (A) following concentration and dialysis (see Methods). Lanes: 1. Hydrophobic interaction chromatography purification of the 27 kDa GFP from cells with the pGLO plasmid. 2. Pooled fractions from the purification protocol. 3. Protein preparation from the pGLO-ATG2 control plasmid. 4. Prestained protein molecular weight markers. (C). Purification of the 74 kDa OrfAB-GFP fusion protein to near homogeneity with the IMPACT system (New England Biolabs) from overexpression of the fused orfAB::GFP genes cloned into the pTWIN2 vector. The eluted protein was subjected to a polishing step on an ion exchange Hi Trap Q sepharose column (GE Healthcare Biosciences). GFP: green fluorescent protein; kDA: kiloDaltons; orf: open reading frame.

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