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.

中文翻译：

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包含α-芋螺毒素TxIB的融合蛋白在大肠杆菌中的表达以及纯化产物中对烟碱型乙酰胆碱受体的选择性保持

烟碱乙酰胆碱受体（nAChRs）是配体门控的离子通道，广泛分布于中枢和周围神经系统。α6β2* nAChR是重要的亚型，与尼古丁成瘾和运动障碍等密切相关。具有16个氨基酸残基的α-芋螺毒素TxIB专门针对α6β2* nAChR，对其他nAChR亚型没有明显影响。然而，TxIB的化学合成是昂贵的，并且从锥蜗牛提取的天然TxIB的量是有限的。在本研究中，我们尝试基于重组表达在大肠杆菌（E. coli）中的生物学方法获得TxIB 。将编码TxIB成熟肽的合成基因插入pET-31b（+）载体中，并转化到大肠杆菌中BLR（DE3）pLysS菌株进行表达。重组融合蛋白KSI‐TxIB‐His ₆（KSI，酮固醇异构酶）成功表达为大肠杆菌中的包涵体。经Ni-NTA亲和色谱柱纯化，并用溴化氰（CNBr）裂解，以释放重组α-芋螺毒素TxIB（rTxIB）。然后，rTxIB通过反相高效液相色谱（RP-HPLC）进行纯化，并通过电喷雾电离质谱（ESI-MS）进行鉴定。rTxIB的药理活性通过电生理学方法进行评估。结果表明，它保留了约50％的效能，但更重要的是，具有与天然芋螺毒素相同的选择性，这可以在不改变其效能的前提下，以低成本低成本提供小批量生产的另一种方法。 。