Usage of Cell-Free Protein Synthesis in Post-Translational Modification of μ-Conopeptide PIIIA

The post-translational modifications of conopeptides are the most complicated to date and well-known closely related activity conopeptides. hydroxylation proline in affects folding, structure, biological activity, prolyl 4 hydroxylase has been characterized Conus literatus. However, machinery is still unclear. In order address mechanism μ-PIIIA, three recombinant plasmids encoding different hybrid precursors μ-PIIIA were constructed crossly combined with protein disulfide isomerase, hydroxylase, glutaminyl cyclase a continuous exchange cell-free system. findings showed that might recognize propeptide achieve proline, while cyclization glutamate was also formed. Additionally, Escherichia coli, co-expression plasmid precursor containing pro mature regions used validate Surprisingly, addition two hydroxyproline residues one pyroglutamyl residue, bridges formed using Trx as fusion tag, yield peptide approximately 20 mg/L. results electrophysiology analysis indicated without C-terminal amidate inhibited current hNaV1.4 939 nM IC50. Our work solved issue it challenging quickly generate post-translationally modified vitro. This first study demonstrate catalyzes through recognition will provide new way for synthesizing multi-modified pharmacological activity.