Figure 2

Purification of recombinant CNPase. A. SDS-PAGE from a Ni-TED purification of a C-terminal CNPase construct. 1, soluble lysate; 2, flow-through; 3-7, washes; 8, marker; 9-15, imidazole elutions. The calculated size for the recombinant protein was 26.7 kDa. B. SDS-PAGE from a Ni-TED purification of a full-length CNPase construct. 1, marker; 2, soluble lysate; 3, flow-through; 4, wash; 5-7, elutions. The calculated size of the recombinant protein was 45.2 kDa. The gels in A and B were stained with PageBlue Protein Staining Solution (Fermentas). The molecular weight marker in both gels is the PageRuler Prestained Protein Ladder, with sizes (from bottom to top) 10, 15, 25, 35, 40, 55, 70, 100, and 130 kDa. C. Size exclusion chromatograms (on a HiLoad 16/60 Superdex 200 column) for different CNPase constructs. The black line is a chromatogram for a full-length CNPase (construct 2-1), the red line for the catalytic domain containing the C-terminal tail (construct 6-2), and the blue line for the catalytic domain without the C-terminal tail (construct 6-1). The absorbance of the catalytic domain without the C-terminal tail was scaled to 10% for it to be in comparable scale. Note the dimer seen in the presence of the C-terminal tail. The elution volumes of molecular weight markers (in kDa) are indicated above the graph.