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.