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 Nav1.7 at an IC50 of 640 nmole/L, similar to that of the natural huwentoxin-I, which is 630 nmole/L.

Recombinant Expression of Margatoxin and Agitoxin-2 in Pichia pastoris: An Efficient Method for Production of KV1.3 Channel Blockers

“…There are three major strategies for obtaining venom peptides: classical biochemical isolation from venom, chemical synthesis, and recombinant production in a heterologous system [19]. Since most venomous animals are very small, bioassay-guided isolation from venom usually produces only a limited amount of native peptide, thus precluding detailed SAR studies.…”

“…The most common host for heterologous expression of venom peptides is E. coli [46], [47] but this often necessitates oxidative refolding of the peptides [48] as the redox environment of the E. coli cytoplasm is not favorable for disulfide-bond formation [16]. Higher yields of correctly folded toxin can often be obtained by exporting the peptides into periplasm of E. coli , where the cellular machinery for disulfide-bond formation is located [17]–[19]; however, the overall yield of recombinant peptide is typically less than 5 mg/liter. Recombinant expression of disulfide-rich peptides in eukaryotic expression systems has also been reported.…”

“…Recombinant production of K V 1.3 blockers in the cytoplasm of E. coli generally necessitates oxidative refolding of the peptides in order to form their native disulfide architecture [16]. An alternative approach that generally avoids the need for refolding is expression of peptides in the periplasm of E. coli [17]–[19] but this often results in low yields. Thus, it would be highly beneficial to develop an efficient and cost-effective method for production of these potent ion channel modulators.…”

Recombinant Expression and In Vitro Characterisation of Active Huwentoxin-IV

“…Alternatively, the ICK-fusion protein can be directed to the periplasm where the oxidative environment allows for disulfide bond formation [10]. Successful ICK secretion using fusion to barnase or a flagellar protein has been reported [13,16].…”

Tailorable nanocarrier emulsion for drug delivery

“…The bio-production of GAM1, which is molecularly equivalent to AM1, requires this small peptide to be produced first as a recombinant protein followed by downstream removal of its high molecular weight fusion tag (Kaar et al, 2009). This is a common procedure in small peptide production in a biofactory system in order to 31 avoid issues related to low expression rates and cytotoxicity to the host system (Anangi et al, 2012, Meng et al, 2011a. However, it is not an economical or sustainable choice as the removal of the large fusion tag not only complicated the production process, but also generates a large amount of waste.…”