Expression and purification of recombinant SjANX A13

Primers of SjANX A13 were designed based on its nucleotide sequence (Genbank no: {"type":"entrez-nucleotide","attrs":{"text":"FN315080.1","term_id":"226471461","term_text":"FN315080.1"}}FN315080.1) using the corresponding restriction enzyme sites of BamHI and XhoI at the N-terminus and C-terminus, respectively. The verified SjANX A13 cDNA fragment was amplified and ligated into the expression vector pET-28a(+) (Novagen, Germany). Products were transformed into Escherichia coli BL21 (DE3) cells (Invitrogen, USA), and recombinant clones were obtained by antibiotic selection. The recombinant proteins were overexpressed in the presence of isopropyl-b-d-thiogalactopyranoside (IPTG). Transformed cells were grown in Luria broth (LB) with 1 mg/mL kanamycin at 37 °C until OD₆₀₀nm = 0.7, and IPTG was added to the culture at a final concentration of 1 mM. After 8 h induction, cells were harvested and the expression of recombinant protein was analyzed by SDS-PAGE. The histidine-tagged fusion recombinant protein was then purified from E. coli lysates by metal affinity chromatography using Ni-NTA His·Bind Resin Chromatography according to the manufacturer’s instructions (Novagen, China). The purified recombinant SjANX A13 (rSjANX A13) protein was analyzed by SDS-PAGE and the protein concentration was measured by a bicinchoninic acid (BCA) assay kit (Yeasen, China). The primers used in this study are listed in Table 3.

Primer sequences used to amplify SjAnnexin A13 gene