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| Codon Optimization | The second PCR fragment was amplified from a plasmid containing a synthetic recodonized pfhp1 sequence (pUC57-re-pfhp1) (GenScript™) (see Supplementary Fig.... The pD-HP1–3M plasmid was constructed by Gibson assembly joining four PCR fragments encoding (1) the 5′ HR spanning bps +88 to +798 of the pfhp1 coding sequence ending with a STOP codon followed by the 103 bp sera2 intron:loxP element and bps +88 to +384 of the recodonised pfhp1 sequence amplified from the pD-PfHP1-Control plasmid using primers F158 and R168, the latter of which introduces the S122/125A mutations into PfHP1; (2) a fragment spanning bps +367 to +798 of the recodonised pfhp1 sequence amplified from pUC57-re-pfhp1 (GenScript™) using primers F91 and R165, the former of which introduces the S125/129A mutations into PfHP1; (3) the g fp coding sequence ending with a STOP codon followed by the 3′ HR amplified from the pFdon-C-loxP-g250 vector (see Supplementary Methods) using primers F162 and R163; and (4) the pD plasmid backbone amplified from pUC19 using primers PCRA_F and PCRA_R72. | Get A Quote |
Previous studies in model eukaryotes have demonstrated that phosphorylation of heterochromatin protein 1 (HP1) is important for dynamically regulating its various functions. However, in the malaria parasite Plasmodium falciparum both the function of HP1 phosphorylation and the identity of the protein kinases targeting HP1 are still elusive. In order to functionally analyze phosphorylation of P. falciparum HP1 (PfHP1), we first mapped PfHP1 phosphorylation sites by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of native PfHP1, which identified motifs from which potential kinases could be predicted; in particular, several phosphorylated residues were embedded in motifs rich in acidic residues... More
