Analysis of CC-chemokine binding evasin sequences. (A) CLUSTAL-W alignment of CC-chemokine binding evasins with evasins 1 and 4 (EVA1, EVA4). Peptide sequence prefix indicates the identity, and suffix indicate the tick species as follows: RHISA and RHIPU – Rhipicephalus sanguineus and pulchellus respectively, and AMBPA, AMBCA, AMBMA, AMBTR - Amblyomma parvum, cajennense, maculatum, triste) respectively. Amino acid residues are color coded by physicochemical properties, and consensus sequence presented above the alignment. Evasin 1 H-bonds to CCL3 are shown as “H”. Evasin 1 structural motifs are indicated as yellow arrows (beta-sheet), green bars (alpha-helix), and blue connectors (disulfide bonds), and were taken from the analysis of the evasin 1:CCL3 structure 3FPU provided in PDBSum18. The blue arrow indicates the P13 residue in evasin 1 that targets the disulfide bond in CCL313. Red arrows indicate F14, N88 and W89 residues in evasin 1 that make contact with CCL3. Green arrows indicate E16 and Y19 residues of evasin 4. The purple arrow indicates N19 in evasin 1, which is glycosylated. The conserved glycosylation sites at the N-terminus predicted using NetNGlyc are boxed in purple. (B) Sequence conservation between CC-chemokine binding evasins and EVA1 and EVA4 peptides. Percent identities between sequences are shown and were calculated by performing BLASTP with default parameters, and are color coded with green indicating high sequence conservation, yellow indicating medium, and orange indicating low conservation. CC-chemokine binding evasins are arranged according to the sequence-similarity based phylogenetic tree shown on the left of the figure. (C) Expression and purification of P991_AMBCA. Colloidal Coomassie stained gels of proteins fractionated by SDS-PAGE. Top panel: Elutions from nickel affinity column, lanes 3–8. Molecular weight ladder (kDa) lane 1. His-tagged P991_AMBCA has a predicted MW including tag of 15.7 kDa. Bottom panel: Fractions collected from size exclusion column chromatography of the pooled material obtained from the nickel affinity column.