Cloning, expression and purification of sPLA2 protein

Total RNA isolated from flax leaves was used for cDNA synthesis using SuperScript™ III First-Strand Synthesis supermix following the manufacturer’s instructions (Life technologies Corporation). Sequence of flax sPLA₂ isoform LusPLA ₂ I (Genbank accession: {"type":"entrez-nucleotide","attrs":{"text":"KU361324","term_id":"1042177905","term_text":"KU361324"}}KU361324) and LusPLA ₂ II (Genbank accession: {"type":"entrez-nucleotide","attrs":{"text":"KU361324","term_id":"1042177905","term_text":"KU361324"}}KU361324) were amplified using primer pairs listed in Supplementary Table ^S7. The amplified gene products were cloned in Gateway entry vector pENTR/SD/D/TOPO (Life technologies Corporation) as per manufacturer’s instructions and authenticity was confirmed by sequencing. For protein expression, the LusPLA ₂ I and LusPLA ₂ II genes were mobilized from entry vector into the destination vector, pET301/CT-DEST (Invitrogen Corporation) to generate the expression clones pET301/CT-DEST harbouring LusPLA ₂ I-6xHis and pET301/CT-DEST harbouring LusPLA ₂ II-6xHis as per manufacturer’s instructions.

The E.coli (CodonPlus (DE3)-RIPL cells (Agilent Technologies) cells harbouring the LusPLA ₂ I- 6xHis and LusPLA ₂ II-6xHis recombinant plasmid were grown at 37°C in LB medium containing 100 µg/ml carbenicillin until OD₆₀₀ = 0.6 and induced with 0.4 mM IPTG (isopropyl-β-D-thiogalactopyranoside) for 4 h. The cells were harvested by centrifugation at 10,000 rpm, 4°C for 10 min and lysed by re-suspending in native lysis buffer containing lysozyme and sonicated at 24–25% amplitude 30 sec ON/OFF cycle on ice for 12–15 minutes ON condition. The recombinant proteins were purified by using Ni-NTA affinity chromatography by using QIAexpress® Ni-NTA Fast Start kit by following manufacturer’s instructions. The purity and yield of recombinant protein were analysed by SDS-PAGE and protein content was determined by Bradford’s method⁶².