Supplementary Material

Supplementary Table. List of cysteine protease genes used for the phylogenetic analysis in Figure 1. (Access here)

Supplementary Figure 1. Structure of cysteine protease from poultry red mites (PRMs, Dermanyssus gallinae) (Deg-CPR-1) and recombinant proteins. (A) The nucleotide and amino acid sequences of Deg-CPR-1 from PRMs collected in Japan. Deg-CPR-1 has the signal peptides at positions 1–18 (dashed line), cathepsin propeptide inhibitor domain at positions 249–305 (grey box), and peptidase domain at positions 335–547 (white box). The black arrow-head indicates an amino acid difference in Deg-CPR-1 between Japanese PRMs and European PRMs at position 535 (aspartic acid in PRMs in Japan; asparagine in PRMs in Europe). The white arrow-heads indicate the predicted active sites for the catalytic residues of the peptidases. (B) The structure of Deg-CPR-1 in the PRMs and recombinant proteins used in this study. For immunisation, the entire recombinant proteins without signal peptides were fused with the histidine tag. For enzyme activity analysis, two recombinant Deg-CPR-1 proteins were used: The peptidase domain fused with histidine-tagged trigger factor (TF), and the whole region without signal peptides fused with histidine-tagged TF. The structure of recombinant protein used in the enzyme activity assay is indicated. (Access here)

Supplementary Figure 2. The expression and purification of cysteine protease from poultry red mites (PRMs, Dermanyssus gallinae) (Deg-CPR-1). The entire recombinant Deg-CPR-1 without the signal peptides, fused with the histidine tag, was expressed and purified for immunisation. The recombinant Deg-CPR-1 was purified from the insoluble inclusion body. The recombinant Deg-CPR-1 was extracted from the insoluble inclusion body and purified using the metal affinity resins. M: Marker (Precision Plus Protein™ All Blue Prestained Protein Standards, Bio-Rad, Hercules, CA, USA). (Access here)

Supplementary Figure 3. The expression and purification of cysteine protease from poultry red mites (PRMs, Dermanyssus gallinae) (Deg-CPR-1) fused with histidine-tagged trigger factor (TF). Two recombinant Deg-CPR-1 proteins fused with histidine-tagged TF were used. The peptidase domain fused with histidine-tagged TF (Deg-CPR-1(PD)-TF), the entire region without signal peptides fused with histidine-tagged TF (Deg-CPR-1(whole)-TF), and TF were expressed and purified for the analysis of enzyme activity. Deg-CPR-1(PD)-TF and TF were purified from the soluble fraction, and Deg-CPR-1(whole)-TF was purified from the insoluble inclusion body. The recombinant proteins were purified using the metal affinity resins. After the elution of recombinant proteins from the resins, the buffer was changed to phosphate-buffered saline by ultrafiltration. M: Marker (Precision Plus Protein™ All Blue Prestained Protein Standards, Bio-Rad). (Access here)

© S. Murata et al., published by EDP Sciences, 2021