Nickel affinity chromatograph

Full-length genes of the four selected proteins were amplified directly from M. hyopneumoniae strain 168 genomic DNA using primer pairs in Table 1. Mycoplasmas use UGA as tryptophan codon while E. coli retain it as a stop codon. In order to mutate TGA to TGG and achieve mycoplasma proteins' heterologous expression, we used specific primers (Table 1) to conduct overlapping PCR for site-directed mutagenesis. Then the products were cloned into pET-28a vector and transformed into E. coli DH5α. After checking the inserts by sequencing, the reconstructed plasmids were transformed into E. coli BL21 (DE3) to express the N-terminal 6×His-tagged recombinant proteins. Obtained proteins were purified by nickel affinity chromatograph (GE Healthcare, Piscataway, NJ, USA), dialyzed in PBS, and concentrated with Amicon Ultra centrifugal filter units (Millipore, Darmstadt, Germany). Protein concentration was determined by BCA Protein Assay Kit (Beyotime, Nanjing, China), and the purity was verified on 12% Coomassie-stained SDS-PAGE gels.

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).