Figure 3
Binding of purified proteins to TP4-A12I,A15I. (a) Band shift of TP4-A12I,A15I by proteins A, B and D analyzed by 8% horizontal native PAGE and Coomassie blue staining. (b) Pulling down of proteins A, B and D by biotinylated TP4-TP4A12I,A15I immobilized on Streptavidin-conjugated beads. (c, d) Biolayer interferometry analysis of protein A, B, and D. The protein D showed potent and reversible binding to biotinylated TP4-A12I,A15I (c), but no substantial binding was observed in protein A and B (d). Heavy lines represent the experimental data, dotted lines represent the global-fitted data for analysis. (e) Inhibition of bactericidal activity of TP4 to E. coli (106 cfu/ml) by protein D not by protein A or B. (f) Decrease of inhibitory effect of FBS on TP4′s antimicrobial activity by the depletion of protein D with TP4-A12I,A15I immobilized beads. (g) FPLC S12 gel filtration chromatography of proteins A/D mixture in the presence of TP4-A12I,A15I. (h, i) Analysis of column eluates as shown in figure g by 15% reduced (h) and non-reducing (i) SDS-PAGE. TP4-A12I,A15I-containing fractions are shown in box.
