Site-Directed Mutagenesis
To confirm if various mutations at residue 172 of tPvPGIP2_5-8 enhance
PG interaction, a saturated site-directed mutagenesis was performed. A
modified version of splicing by overlap extension (SOE) was done. Five
oligonucleotides, listed in Table S3, were annealed together to create
tPvPGIP2_5-8 mutants with residue 172 replaced in the first
oligonucleotide. Each PCR reaction contained 20µL of DI water, 2.5µL of
Expand polymerase, 0.5µL of 10mM dNTPs, 0.3µL of each oligonucleotide,
and 0.3µL of Expand polymerase. Afterwards, an additional 0.3µL of the
first and last oligonucleotide were added, along with 0.3µL dNTPs and
0.2µL Expand polymerase for another round of PCR. The product was run on
a 0.8% agarose gel, then gel purified to retrieve the DNA. The
assembled DNA fragment was then inserted to the corresponding vector
containing the AD-Gal domain through Gibson Assembly.