Optimal Cleavage and Oxidative Folding of α-Conotoxin TxIB as a Therapeutic Candidate Peptide

Abstract

Alpha6beta2 nicotinic acetylcholine receptors (nAChRs) are potential therapeutic targets for the treatment of several neuropsychiatric diseases, including addiction and Parkinson’s disease. Alpha-conotoxin (α-CTx) TxIB is a uniquely selective ligand, which blocks α6/α3β2β3 nAChRs only, but does not block the other subtypes. Therefore, α-CTx TxIB is a valuable therapeutic candidate peptide. Synthesizing enough α-CTx TxIB with high yield production is required for conducting wide-range testing of its potential medicinal applications. The current study optimized the cleavage of synthesized α-CTx TxIB resin-bounded peptide and folding of the cleaved linear peptide. Key parameters influencing cleavage and oxidative folding of α-CTx TxIB were examined, such as buffer, redox agents, pH, salt, co-solvent and temperature. Twelve conditions were used for cleavage optimization. Fifty-four kinds of one-step oxidative solution were used to assess their effects on each α-CTx TxIB isomers’ yield. The result indicated that co-solvent choices were particularly important. Completely oxidative folding of globular isomer was achieved when the NH4HCO3 or Tris-HCl folding buffer at 4 °C contained 40% of co-solvent DMSO, and GSH:GSSG (2:1) or GSH only with pH 8~8.7.

Expression in Escherichia coli of fusion protein comprising α‐conotoxin TxIB and preservation of selectivity to nicotinic acetylcholine receptors in the purified product

“…But chemical synthesis of TxIB is expensive; the yield of native TxIB synthesized by a two-step oxidative folding method is only about 30%. [19] However, the yield of rTxIB by biosynthesis in this work could be several even dozens of times higher than chemical synthesis, which decreased the peptide production cost. So this biosynthesis method could be considered as an alternative way to obtain abundant peptides.…”

“…[19] However, the yield of rTxIB by biosynthesis in this work could be several even dozens of times higher than chemical synthesis, which decreased the peptide production cost. As the number of C. textile is decreasing and the quantity of TxIB naturally secreted is very limited, chemical synthesis is the major method of TxIB production at present.…”

“…[22] Especially for α-CTx TxIB isomers, only the globular TxIB was fully active on α6/α3β2β3 nAChR, but the rest two isomers, ribbon and bead TxIB, were inactive on α6/α3β2β3 nAChR at all. [19] 4 | DISCUSSION At least 1,000,000 unique kinds of conopeptides exist in the world. Therefore, we may infer that active rTxIB should have two disulfide bonds of globular isomer with Cys I-III and Cys II-IV connectivities.…”

“…After the linear peptide of TxIB was synthesized and oxidated by a two-step oxidation process artificially, the native TxIB was set as positive control. [19]…”

“…In addition, the "globular" TxIB was the main product by one-step random oxidation folding, while the "bead" isomer was not detected. [19]…”