Figure 1.

ABD–Dox synthesis. A) Amino acid sequence of ABD. B) Schematic illustration of the design and synthesis steps. ELP was used as a purification tag for ABD purification from bacteria and was removed following drug conjugation using sortase A. The KEKE peptide was included at the N-terminus to disrupt micelle self-assembly upon Dox conjugation, and to enable the subsequent sortase A cleavage of the ELP from the ABD–Dox conjugate. C) SDS-PAGE analysis confirmed successful purification of KEKE–ELP₁₆₀–srt–ABD–(GGC)₄ and (His)₆–sortase A–ELP₄₀ with theoretical molecular weights of 69.4 and 34.4 kDa, respectively. D) Hydrodynamic radius of ELP₁₆₀–srt–ABD–Dox conjugates was measured with DLS. ELP₁₆₀–srt–ABD–Dox conjugates without N-terminal KEKE segment self-assembled into micelles with R_h of 40–50 nm, whereas conjugates containing the N-terminal KEKE segment were mostly (≈90%) unimers with R_h < 10 nm.