Cloning and Mutagenesis of PfGSK3 Protein

The vector with N-terminally His-tagged PfGSK3 was generated by PCR amplification of the GSK3 coding sequence from P. falciparum cDNA followed by Ligation Independent Cloning into HindIII/KpnI-cleaved plasmid pOPIN F [48 (link)] using the In-Fusion HD EcoDry Cloning Kit (Takara Clontech) according to the manufacturer's instructions. The mutants S226A, Y229A and S226A/Y229A were generated by overlap extension PCR amplification from the original vector and Ligation Independent Cloning as described above. The wild-type protein and the mutant K96A cloned in pET28a vector were ordered from GenScript. The N-terminally truncated constructs were cloned by amplifying the sequence from the original vector and subcloning into BsaI-cleaved plasmid pNIC28_Bsa4 by SLiCE cloning [49 (link)].