To generate endogenously tagged small gatekeeper kinases, the putative kinase-coding genes were amplified from genomic DNA by PCR and cloned into the pKS_3HA_Neo vector [29 (link)]. Primer sequences and restriction enzymes are shown in S1 Table . PCR amplifications were performed using iProof DNA polymerase (Bio-Rad). Typically, an amplicon of ~ I kb in length that lacked the start codon was cloned in frame to a C-terminal triple-hemagglutinin epitope tag (3xHA) into the pKS_3HA_Neo plasmid. The plasmids were linearized by the enzyme reported in S1 Table and ~5 μg of DNA was used to transform wild-type Giardia. Transformants were selected with G418 at 40 μg/mL.
For protein expression, the complete coding region of each protein kinase was PCR amplified from Giardia genomic DNA. PCR amplicons were cloned into the ligation independent cloning (LIC) site of (MBP)-AVA0421 expression vector and validated by sequencing [30 (link)]. Recombinant proteins were expressed in E. coli BL21 (DE3), Invitrogen, Carlsbad, CA) using Studier auto-induction protocols at 20°C [31 (link)]. Recombinant protein purifications were performed as previously described [26 (link)].
For protein expression, the complete coding region of each protein kinase was PCR amplified from Giardia genomic DNA. PCR amplicons were cloned into the ligation independent cloning (LIC) site of (MBP)-AVA0421 expression vector and validated by sequencing [30 (link)]. Recombinant proteins were expressed in E. coli BL21 (DE3), Invitrogen, Carlsbad, CA) using Studier auto-induction protocols at 20°C [31 (link)]. Recombinant protein purifications were performed as previously described [26 (link)].
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