Cloning, expression and purification of PaTrm5a

The wild type (WT), full-length PaTrm5a gene was chemically synthesized (Genewiz, Suzhou, China) and PCR-amplified. After the double digestion by the restriction enzymes NdeI and XhoI, the digested PCR product was inserted into a modified pET-28a (+) vector, where the thrombin recognition site (LVPRGS) in pET-28a (+) was replaced by the PreScission protease cleavage site LEVLFQGP. For the FRET experiments, several rounds of PaTrm5a mutagenesis were carried out for fluorophore-labeling: in the first round, the unwanted cysteines in WT PaTrm5a were mutated using three consecutive QuikChange (Stratagene) PCRs to produce the triple mutant C301S/C308S/C326S with the QuikChange primers (including the sense and antisense primers) for the C301S, C308S and C326S mutations respectively (Supplementary Table S2); in the second round, two cysteines were introduced into the triple mutant using two more QuikChange PCRs with the primers for the V21C and K314C mutations to produce the mutant M1 (with the mutations V21C/C301S/C308S/K314C/C326S); in the last round, the mutant M2 was generated from M1 using two PCRs through the SCGI-to-ECNL and VRCVS-to-KRCVK primers. All the amplifying and QuikChange primers used in this study were listed in Supplementary Table S2.

The plasmids of PaTrm5a and mutants were transformed into the Escherichia coli strain BL21 (DE3) cells for overexpression. The harvested cells were lysed by sonication. The cell debris was removed by centrifugation, and the supernatant was then applied onto a Ni-NTA affinity column (Qiagen). In order to remove the endogenous nucleic acid bound to the recombinant PaTrm5a, the eluted fractions were collected and treated overnight with 0.08 mg/mL RNase A in the presence of 5 mM MgCl₂. The protein was further subjected to affinity purification by a heparin column (GE Healthcare) using a NaCl gradient, and PaTrm5a was eluted at ~500 mM NaCl. The purified protein was pooled, dialysed in a buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl and 1 mM DTT (without DTT for the M1 mutant) and stored after being flash-frozen.