Baculovirus insect expression system

To identify and locate the complement C1q binding site on TsPmy, the full-length T. spiralis paramyosin (TsPmy1-885aa, NCBI accession numbers for nucleotides sequence: EF429310.1 and protein sequence: ABO09862.1) was expressed as recombinant protein using a baculovirus insect expression system (Invitrogen, Carlsbad, CA, USA), and the subsequent fragments of TsPmy, with 30 amino acids overlapped (TsPmy1-315aa, TsPmy286-600aa, TsPmy571-885aa), were sub-cloned and expressed in an E. coli expression system (Novagen, Merck, Darmstadt, Germany) as described in [4 (link)]. Further fragmenting expression on the C1q-binding fragments of TsPmy1-315aa (TsPmy1-125aa, 96-220aa, 191-315aa) and TsPmy191-315aa (TsPmy191-245aa, 226-280aa, 261-315aa) was performed in E. coli. All recombinant proteins were expressed with 6-histidine tag and purified by nickel affinity chromatograph (GE Healthcare, Boston, MA, USA).

Trichinella spiralis paramyosin (GenBank# ABO09862.1) was expressed as recombinant protein (rTsPmy) in the baculovirus insect expression system (Invitrogen, Carlsbad, CA, United States). The rTsPmy with hexahistidine-tag at the C-terminus was expressed as partially soluble protein. After being denatured with guanidine hydrochloride, rTsPmy was purified by Ni-affinity chromatography (GE Healthcare, Boston, MA, United States) followed by a refolding process using a Protein Refolding Kit (Novagen/Merck KGaA, Darmstadt, Germany). The refolded soluble rTsPmy was characterized by SDS-PAGE and could be recognized by anti-rTsPmy sera by western blot.