Identification of Four Entamoeba histolytica Organellar DNA Polymerases of the Family B and Cellular Localization of the Ehodp1 Gene and EhODP1 Protein
Figure 6
Expression and identification of rEhODP1-168 polypeptide. (a) rEhODP1-168 polypeptide production was 1 mM IPTG induced in bacteria, and proteins were separated by 15% SDS-PAGE. Gel was Coomassie Brilliant Blue stained. Lane 1, protein molecular weight markers. Lane 2, uninduced bacteria. Lane 3, induced bacteria. (b) The induced recombinant protein was purified by IMAC using denaturing conditions. Fractions were analyzed by 15% SDS-PAGE and the gel was Coomassie Blue stained. Lane 1, protein molecular weight standards. Lane 2, total extract from IPTG-induced bacteria before the affinity chromatography. Lane 3, unbound proteins to the Ni2+-NTA-agarose column. Lane 4, proteins eluted with buffer D. Lane 5, proteins eluted with buffer E (see Materials and Methods). (c) Purified proteins were separated by 15% SDS-PAGE, transferred to a nitrocellulose membrane and stained with Ponceau Red. Lane 1, prestained protein molecular weight markers. Lane 2, proteins in fraction D. (d) Western blot of proteins shown in lane 2 in (c), immunodetected with monoclonal anti-6His tag antibodies. (e) 2D gel electrophoresis of proteins eluted in fraction D from the Ni2+-NTA-agarose column. Proteins were stained with Coomassie Brilliant Blue. (f) Amino acid sequence of the expected rEhODP1-168 that corresponds to the spot shown in (e) (arrow). Underlined are the peptides identified by MALDI-TOF mass spectrometry. Arrows indicate the expected rEhODP1-168 polypeptide. Arrowheads show an induced polypeptide that was copurified with rEhODP1-168.
