Functional expression of spider neurotoxic peptide huwentoxin-I in E. coli.
Functional expression of spider neurotoxic peptide huwentoxin-I in E. coli.
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DOI：
10.1371/journal.pone.0021608
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Zhang DY
中科院分区：
文献类型：
--
作者：
Meng E;Cai TF;Li WY;Zhang H;Liu YB;Peng K;Liang S;Zhang DY
利用构建的蜘蛛毒腺cDNA文库，用PCR方法扩增了从中华鸟爪蛛（Ornithoctonus huwena）毒液中分离得到的神经毒肽huwentoxin-I的编码序列。将克隆片段插入表达载体pET-40b中，转化大肠杆菌BL21 （DE3）。在没有IPTG的情况下，可溶性融合蛋白二硫化物交换蛋白(DsbC)-huwentoxin-I在大肠杆菌的周质中被自动诱导表达。表达的融合蛋白经Ni-NTA柱部分纯化后，用肠激酶消化释放异表达的huwentoxin-I，然后用RP-HPLC进一步纯化。所得肽进行凝胶电泳和质谱分析。异表达的huwentoxin-I的分子量为3750.69，与从蜘蛛毒液中分离的肽的天然形态相同。利用全细胞膜片钳法进一步分析异表达胡文毒素i的生理特性。异表达的huwentoxin-I能够阻断人Nav1.7产生的电流，IC50为640 nmol /L，与天然的huwentoxin-I的IC50相似，为630 nmol /L。
The coding sequence of huwentoxin-I, a neurotoxic peptide isolated from the venom of the Chinese spider Ornithoctonus huwena, was amplified by PCR using the cDNA library constructed from the spider venom glands. The cloned fragment was inserted into the expression vector pET-40b and transformed into the E. coli strain BL21 (DE3). The expression of a soluble fusion protein, disulfide interchange protein (DsbC)-huwentoxin-I, was auto-induced in the periplasm of E. coli in the absence of IPTG. After partial purification using a Ni-NTA column, the expressed fusion protein was digested using enterokinase to release heteroexpressed huwentoxin-I and was further purified using RP-HPLC. The resulting peptide was subjected to gel electrophoresis and mass spectrometry analysis. The molecular weight of the heteroexpressed huwentoxin-I was 3750.69, which is identical to that of the natural form of the peptide isolated from spider venom. The physiological properties of the heteroexpressed huwentoxin-I were further analyzed using a whole-cell patch clamp assay. The heteroexpressed huwentoxin-I was able to block currents generated by human Nav1.7 at an IC50 of 640 nmole/L, similar to that of the natural huwentoxin-I, which is 630 nmole/L.
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影响因子：
2.8
作者：
Diao, JB;Lin, Y;Liang, SP
通讯作者：
Liang, SP
影响因子：
1.6
作者：
Ji, WJ;Zhang, XY;An, CC
通讯作者：
An, CC
鞘内注射虎纹鸟毒素-I（一种选择性 N 型钙通道阻滞剂）在清醒大鼠福尔马林试验中的镇痛作用
影响因子：
2.8
作者：
Chen, JQ;Zhang, YQ;Liang, SP
通讯作者：
Liang, SP
影响因子：
4.8
作者：
Maggio, F;King, GF
通讯作者：
King, GF
DOI：
10.1023/a:1026314722607
发表时间：
1997-08-01
期刊：
JOURNAL OF PROTEIN CHEMISTRY
影响因子：
--
作者：
Qu, YX;Liang, SP;Gu, XC
通讯作者：
Gu, XC