Recombinant Sortilin Protein Production

The protein-coding region of the sortilin gene (gene number: TGGT1_290160) was obtained from DNA isolated from T. gondii RH tachyzoites by PCR using gene-specific primers (Additional file 1: Table. S1). The amplicon was cloned into pET-28a and pGEX-4T-1 vectors (Invitrogen, Carlsbad, CA, USA). The two recombinant plasmids were expressed in Escherichia coli BL21 (DE3) cells (TransGen Biotech, Beijing, China). His- and GST-tagged recombinant proteins were obtained by affinity purification, as described previously [33 (link)]. Purified recombinant proteins were detected by Western blotting using specific antibodies against HIS and GST tags (Additional file 1: Fig. S2).
Four subdomain coding regions, named sortilin-N (1–166 aa), Vps10 (160–667 aa), sortilin-C (665–848 aa), and sortilin-M (880–1033 aa), were obtained using specific primers (Additional file 1: Table. S1) as described above. The four amplified gene fragments were cloned into the pGEX-4T-1 vector and expressed in E. coli BL21 cells (DE3). Finally, recombinant GST proteins were obtained by affinity purification using Glutathione Sepharose 4 B (Cytiva, Logan, UT, USA) (Additional file 1: Fig. S3A, B), as previously described [33 (link)].