Pet28a tev

The chimeric protein-codifying gene sequence was commercially synthetized as having 1107 bp and it was cloned into prokaryotic expression vector pET28a-TEV (Genscript^®, Piscataway, NJ, USA). The construct was transformed into Escherichia coli BL-21 strain cells, and the protein was expressed by addition of 0.2 mM isopropyl-β-d-thiogalactopyranoside (IPTG; Promega, Madison, WI, USA) for 2 h at 37 °C in 1 L of LB medium (with yield of 36 mg/L of culture). Next, bacteria were centrifuged at 3000× g for 10 min at 4 °C and suspended in lysis buffer (20 mM Tris-HCL, pH 8.0; 0.5 M NaCl; 5 mM imidazole; 8 M urea; 1 mM β-mercaptoetanol), followed by six cycles of ultrasonication for 30 s each (at 90 Hz). Cellular debris was removed after 5000× g centrifugation for 15 min at 4 °C and the supernatant was collected. The N4S11-SC2 protein was purified on a HisTrap HP affinity column (GE Healthcare Life Sciences, Chicago, IL, USA) connected to an AKTA system. Inclusion bodies were solubilized in 2 M urea buffer. A 12.5% (w/v) SDS-PAGE was done to evaluate the purity of the recombinant protein. A Page Ruler broad range unstained protein ladder (Thermo Fisher Scientific Baltics, Vilnius, Lithuania) was used.

The vectors to express Vs NnlA and H73A Vs NnlA were previously reported [21 (link)]. Pd, Mr, Ms, Ps, and Pj nnla genes were synthesized as E. coli codon-optimized constructs and cloned into the NdeI and XhoI restriction sites of pET-28a(+)-TEV by GenScript.
For protein expression, plasmids were electroporated into E. coli BL21(DE3) cells and protein was expressed and purified by immobilized metal affinity chromatography (IMAC) as previously described for Vs NnlA [21 (link)]. The only modification was that plasmids using pET-28a(+)-TEV required 50 µg/mL kanamycin instead of 100 µg/mL ampicillin. IMAC purified and concentrated protein were exchanged into 100 mM tricine, 100 mM NaCl buffer at pH 7.5 and stored at −60 °C.