Epitope Tagging of PfSUB2

<div><p>(A) Schematic depiction of integration plasmids pHH1-T996HA3 and pHH1-T996w, and single-crossover homologous recombination events (for clarity shown only for pHH1-T996HA3). Integration reconstitutes a full-length <i>pfsub2</i> gene either with or without a triple HA tag at the 3′ end. Correct transcription termination and polyadenylation of this gene is regulated by the presence of the <i>P. berghei</i> dihydrofolate reductase 3′ UTR (PbDT 3′ UTR). <i>hdhfr,</i> human dihydrofolate reductase, conferring resistance to the antifolate WR99210. S, indicates the ScaI sites. P indicates the position and size of the probe used for Southern analysis.</p><p>(B) Southern blots of ScaI-digested input plasmid pHH1-T996HA3 or genomic DNA from PfSUB2w clones F7 and D8, PfSUB2HA clones 10E and 2D, and wild-type D10 parasites. The position of the genomic 3.4-kb fragment derived from the wild-type (wt) <i>pfsub2</i> locus is arrowed. Its disappearance from all the transgenic clones indicates disruption of this locus, whilst the appearance of the additional species at 5.8 kb and 4.4 kb indicates integration as predicted. The fragments seen at the position of full-length input plasmid derive from integration of more than one copy of the entire plasmid, as described in the text.</p><p>(C) Western blot of transgenic clones using anti-HA mAb 3F10 (identical results were obtained using anti-HA mAb 12CA5; not shown) or anti-RAP2 mAb H5, used as a loading control. Identities of the authentic PfSUB2 species are indicated, and the 55-kDa PfSUB2 degradation product (the abundance of which varied widely between experiments, being most prominent in non-ionic detergent extracts incubated for prolonged periods) is marked with an asterisk. Note that the predicted molecular mass of an HA-tagged minimal PfSUB2 catalytic domain (extending from the catalytic Asp755 to the end of the C-terminal tag) is approximately 70 kDa. Molecular masses are in kDa.</p>