Total chemical synthesis of pentameric cholera toxin subunit B

<i>Vibrio cholerae</i> annually infects millions worldwide, causing intense and life-threatening diarrheal disease. The pentameric cholera toxin subunit B (CtxB) binds to the ganglioside GM1 on epithelial cells, initiating endocytosis and downstream toxicity. We present the total chemical protein synthesis of L-CtxB and mirror-image D-CtxB using a three-segment N-to-C native chemical ligation approach. Oxidative folding produced the desired 62 kDa pentameric proteins, with L-CtxB capable of binding native GM1 ligand. Synthetic D-CtxB is now ready to serve as a target in mirror-image phage display, representing a significant milestone for the discovery of toxin-neutralizing D-peptides to treat and prevent cholera.