Fig. 2

BBB homing peptide derived from OspA of Borrelia and its fusion with Cx₇C. (A) Organization of domains in OspA monomer. Tick gut epithelial cell binding domain 1 (TGE1) - green, TGE2 - orange, TGE3 – red, endothelial cell binding domain – blue and magenta. N-terminal fragment of the endothelial cell binding domain is in blue (comprised of two beta sheets 10 and 11), while C terminal part in magenta is comprised on 5 beta sheets (12 to 16). (B) Time dependent translocation of OspA derived peptides through BBB model. N terminal fragment of endothelial cell binding domain (O-BBB), C terminal fragment, full endothelial cell binding domain, angiopep-2-Cy5.5, dextran-CF770. ***Significantly lower crossing (two tailed t-test P < 0.05) of designated molecules than O-BBB and angiopep-2. Significant difference between crossing of angiopep-2 and O-BBB was observed (P = 0.0175) at 1st hr. At 2nd hr crossing of O-BBB was matched with angiopep-2, while at 3rd hr significantly higher crossing of O-BBB was evident. Thus, for the fusion of CX₇C, O-BBB was preferred in the study. Error bars represent the standard deviation of triplicate measurements. (C) A graphical representation of the Cx₇C fused O-BBB. Beta sheets forming O-BBB are colored blue, Cx₇C (red loop) is separated from O-BBB by a linker. Yellow dots represent cysteine residues that help to form a loop-like structure. (D) Schematic representation of the construct to produce anti-Borrelia Cx₇C peptide fused with O-BBB. Construct contains 6x histidine tag, O-BBB, GGGS linker, sequence digested by enterokinase protease (Ent), ACxxxxxxxC – 7-mer peptides which showed affinity to Borrelia cell membrane (peptide is are flanked by cysteine residues and alanine at N-terminus), three glycine residues followed by serine and then stop codon.