title: Functional expression of spider neurotoxic peptide huwentoxin-I in E. coli.
abstract: 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 Na(v1.7) at an IC₅₀ of 640 nmole/L, similar to that of the natural huwentoxin-I, which is 630 nmole/L.
authors: Key Laboratory of Protein Chemistry and Developmental Biology of the Ministry of Education, College of Life Sciences, Hunan Normal University, Changsha, Hunan, China.
authors: Er
authors: E
authors: Meng
authors: Tian-Fu
authors: TF
authors: Cai
authors: Wen-Ying
authors: WY
authors: Li
authors: Hui
authors: H
authors: Zhang
authors: Yan-Bo
authors: YB
authors: Liu
authors: Kuan
authors: K
authors: Peng
authors: Songping
authors: S
authors: Liang
authors: Dong-Yi
authors: DY
authors: Zhang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects; D057809:HEK293 Cells; D006801:Humans; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D062556:NAV1.7 Voltage-Gated Sodium Channel; D009498:Neurotoxins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000633:toxicity; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000633:toxicity; D030162:Reptilian Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000633:toxicity; D015222:Sodium Channels / Q000378:metabolism; D013111:Spider Venoms / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000633:toxicity; D013112:Spiders / Q000378:metabolism*
chemical_list: D062556:NAV1.7 Voltage-Gated Sodium Channel; D009498:Neurotoxins; D010455:Peptides; D030162:Reptilian Proteins; C486866:SCN9A protein, human; D015222:Sodium Channels; D013111:Spider Venoms; C083624:huwentoxin I