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Expression in Escherichia coli of fusion protein comprising α-conotoxin TxIB and preservation of selectivity to nicotinic acetylcholine receptors in the purified product.
Chemical Biology & Drug Design 2018 Februrary
Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels, which are widely distributed in the central and peripheral nervous system. The α6β2* nAChR is an important subtype, which is closely associated with nicotine addiction and movement disorders etc. α-conotoxin TxIB with 16-amino acid residues specifically targets α6β2* nAChR with no obvious effect on other nAChR subtypes. However, chemical synthesis of TxIB is expensive, and the quantity of native TxIB extracted from cone snail is limited. In the present study, we attempted to obtain TxIB using biological method based on the recombinant expression in Escherichia coli (E. coli). The synthetic gene encoding mature peptide of TxIB was inserted in pET-31b(+) vector and transformed into E. coli strain BLR(DE3)pLysS for expression. The recombinant fusion protein KSI-TxIB-His6 (KSI, ketosteroid isomerase) was expressed successfully as inclusion body in E. coli, which was purified by Ni-NTA affinity chromatography column and cleaved by cyanogen bromide (CNBr) to release recombinant α-conotoxin TxIB (rTxIB). Then, rTxIB was purified by reverse-phase high-performance liquid chromatography (RP-HPLC) and was identified by electrospray ionization mass spectrometry (ESI-MS). Pharmacological activity of rTxIB was assessed by electrophysiological approaches. The results indicated that it preserved about 50% of potency, but, was even more important, had the same selectivity as the natural conotoxin which may provide an alternative method for quantity production of small peptides with low cost on the premise of not changing their potency.
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