2.6.1 Protein expression and purification
The target GmolCXE proteins were expressed and purified as described in
previous studies (Wei et al., 2019; Zhu et al., 2017). In brief, cDNAs
were amplified using specific primers with enzyme recognition sites
(Table S2). After digestion with restriction enzymes, the target gene
fragments were inserted into expression vector pET30a (+) digested with
the same restriction enzymes. The recombinant plasmids with target
GmolCXE genes were induced in Escherichia coli Transetta (DE3)
cells with 1 mM I isopropyl-β-D-thiogalactoside (IPTG) at 15 ℃ for 12 h.
After sonication and centrifugation, recombinant proteins of GmolCXE1,
14 were mainly present as inclusion bodies, and GmolCXE5, 21 major
expressed in supernatant. The inclusion bodies were dissolved in 6 M
Guanidine Hydrochloride and purified with Tris buffer containing 8 M
urea by Ni ion affinity chromatography (GE Healthcare Biosciences,
Uppsala, Sweden). The supernatant were purified directly by the Ni ion
affinity chromatography Then the purified proteins were refolded by
extensive dialysis against 20 mM Tris-HCl buffer (PH=7.40).