4.2 Generation of PEXEL P2’ mutants and spacer truncation constructs
The expression of Hyp1/ STEVOR/ KAHRP-Nluc-mDH-FL reporters was driven by a bidirectional Plasmodium berghei EF1a promoter that also controlled expression of the blasticidin deaminase drug resistance cassette. The plasmid pEF-Hyp1-Nluc-mDH-FL was derived from plasmid pEF-Hyp1-Nluc-DH-APEX 43. The Hyp1 component of this plasmid contained the first 113aa of Hyp1 (PF3D7_0113300), including the RLLTE PEXEL motif 43. A synthetic murine dihydrofolate reductase (mDH) gene fragment with C-terminal 3x FLAG epitopes (Bioneer Pacific) was ligated into the Nluc-DH-APEX plasmid using SpeI and MluI enzymes to remove the previous mDH-APEX gene cassette.
Generation of the P2’ lysine (lys/K) and alanine (ala/A) mutation of the Hyp1-Nluc-mDH-FL was performed as follows: Hyp1 region was first amplified as two overlapping PCR fragments with the primer pairs 1 & 2 and 3 & 4 (Table S4). The overlap region between these two PCR products contained the mutations indicated above. PCR fragments were then sewn together with primer pair 1 & 4 via overlapping PCR, ligated into the pJET1.2/blunt plasmid (ThermoFisher Scientific). Mutagenesis was confirmed via standard Sanger sequencing (service provided by Monash Micromon Genomics) of the isolated plasmids. The mutant P2’ K/A Hyp1 fragments were released from pJET1.2/blunt using XhoI and NcoI and ligated into pEF-Hyp1-Nluc-mDH-FL to replace the wildtype Hyp1 fragment.
Generation of the Hyp1-Nluc-mDH-FL plasmids with a truncated spacer region between the PEXEL motif and the start of the Nluc gene was carried out as follows: PCRs were performed with primer 1 paired with primer 7, 8 or 9 to produce spacers of 3aa, 13aa and 51aa, respectively (Table S4). Note that primers 7, 8 and 9 produced Hyp1 PCR products that contained one additional amino acid between the last Hyp1 residue and the start Met of Nluc. The PCR products were ligated into pJET1.2/blunt and screened as above.
To express wildtype STEVOR (PF3D7_0200400) and KARHP (PF3D7_0202000) in the Nluc-mDH-FL reporter plasmid, synthetic leader sequences encoding the first 99aa of STEVOR and 105aa of KAHRP containing XhoI andNcoI restriction sites were first obtained as string oligos from GeneART (ThremoFisher Scientific). P2’ lysine mutations for both STEVOR and KAHRP leaders were also synthesised as above. The synthetic DNA sequences were ligated into pJET1.2/blunt and validated by sequencing prior to transfer via XhoI and NcoI sites into the pEF-Hyp1-Nluc-mDH-FL plasmid replacing the Hyp1 sequence.
To produce pEF-STEVOR-Nluc-mDH-FL reporters with truncated spacers, PCRs were performed with primers 10 & 11 and primers 10 & 12 to produce spacer fragments of 3aa, and 13aa respectively, using pEF-STEVOR-Nluc-mDH-FL as a template (Table S5). PCR products were ligated into pJET1.2/blunt and ligated into pEF-Nluc-mDH-FL viaXhoI and NcoI cloning sites to produce gene fusions with only one amino acid between STEVOR/KAHRP leader sequence and Nluc. To produce the pEF-KAHRP-Nluc-mDH-FL with 3aa and 13aa spacers, PCRs were performed with primers 10 & 13 and primers 10 & 14 (Table S4). The PCR products were ligated into the Nluc-mDH-FL reporter as per the STEVOR reporters.