Screening for Voltage-Gated Sodium Channel Interacting Peptides

Meng, Er; Cai, Tianfu; Zhang, Hui; Tang, Si; Li, Mengjie; Li, Wenying; Huang, Pengfei; Li, Kai; Wu, Lei; Zhu, Lingyun; Liu, Long; Peng, Kuan; Dai, Xiandong; Jiang, Hui; Zeng, Xin; Liang, Songping; Zhang, Dongyi

doi:10.1038/srep04569

Cited by 8 publications

(4 citation statements)

References 28 publications

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“…The whole-cell patch-clamp experiment was performed to analyse the electrophysiological activities of rJZTX-III. As demonstrated in Figure 5, 1 mmol/L rJZTX-III could significantly inhibit the current of hNav1.5 expressed in HEK293 cells, which was similar to the effects observed for the natural JZTX-III [15]. This result suggests that rJZTX-III expressed in the cytoplasm of the SHuffle strain through the galactose auto-induction method retains its bio-functional activity.…”

Section: Electrophysiological Properties Analysis Of Rjztx-iiisupporting

confidence: 78%

An efficient strategy for the expression of Jingzhaotoxin-III inEscherichia coli

Meng

Wang

et al. 2017

Biotechnology & Biotechnological Equipment

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Jingzhaotoxin-III (JZTX-III), a 36-residue peptide cardiotoxin containing three pairs of disulphide bonds, has been characterized from the venom of the Chinese tarantula Chilobrachys jingzhao. JZTX-III is a promising target for drug development and clinical application, due to its specific inhibitory effects on the human voltage-gated potassium channel subtype hKv2.1 and sodium channel subtype hNav1.5, which are mainly expressed in the cardiac myocytes. The most direct way to obtain JZTX-III is by extraction from the native venom of the tarantula jingzhao, but the amount is often insufficient to meet research and clinical demands. Therefore, there is need for an efficient expression system that can provide a larger quantity of JZTX-III. In this paper, we utilized a galactose auto-induction system to assist the Escherichia coli strain SHuffle T7 Express to express recombinant JZTX-III, followed by in situ purification on a Ni-nitrilotriacetic acid (Ni-NTA) column. Subsequent experiments were performed to demonstrate the advantages of the galactose autoinduction method and to optimize the incubation conditions. Under the optimal expression conditions, the product of the purified bioactive recombinant JZTX-III reached 12.1 mg/L. Furthermore, it is expected that this expression method can be widely applied to the heterologous expression of other disulphide-bond-rich peptides.

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Section: Electrophysiological Properties Analysis Of Rjztx-iiisupporting

confidence: 78%

An efficient strategy for the expression of Jingzhaotoxin-III inEscherichia coli

Meng

Wang

et al. 2017

Biotechnology & Biotechnological Equipment

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“…Chimaeragenesis is a useful tool for the study of relationships of protein structure and function. For example, by constructing different voltage-gated channel subtypes chimaeras, chimaeragenesis already revealed some important gating properties of the less-characterized sodium channels, such as Na v 1.9 and Na v 1.8 [ 8 , 9 ]. Chimaeragenesis, by which two or more DNA modules could be stitched together, is generally achieved by in vivo [ 10 ] or/and in vitro [ 11 ] recombination or overlap-extension PCR [ 12 , 13 ].…”

Section: Resultsmentioning

confidence: 99%

Mega primer-mediated molecular cloning strategy for chimaeragenesis and long DNA fragment insertion

et al. 2017

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Molecular cloning methods based on primer and overlap-extension PCR are widely used due to their simplicity, reliability, low cost and high efficiency. In this article, an efficient mega primer-mediated (MP) cloning strategy for chimaeragenesis and long DNA fragment insertion is presented. MP cloning is a seamless, restriction/ligation-independent method that requires only three steps: (i) the first PCR for mega primer generation; (ii) the second PCR for exponential amplification mediated by the mega primers and (iii) DpnI digestion and transformation. Most importantly, for chimaeragenesis, genes can be assembled and constructed into the plasmid vector in a single PCR step. By employing this strategy, we successfully inserted four DNA fragments (approximately 500 bp each) into the same vector simultaneously. In conclusion, the strategy proved to be a simple and efficient tool for seamless cloning.

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“…Nevertheless, other interactions might not be detected in YTH assay because of the lack of chaperones, assembly factors, post-translational modifications, or other effects 43 . In addition, the YTH assay may produce false positives if (1) the two proteins are not expressed in the same tissue or at the same time or if (2) the prey protein has a self-activating function 43 44 .…”

Section: Discussionmentioning

confidence: 99%

Integrative proteomics to understand the transmission mechanism of Barley yellow dwarf virus-GPV by its insect vector Rhopalosiphum padi

Wang

Liu

et al. 2015

Sci Rep

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Barley yellow dwarf virus-GPV (BYDV-GPV) is transmitted by Rhopalosiphum padi and Schizaphis graminum in a persistent nonpropagative manner. To improve our understanding of its transmission mechanism by aphid vectors, we used two approaches, isobaric tags for relative and absolute quantitation (iTRAQ) and yeast two-hybrid (YTH) system, to identify proteins in R. padi that may interact with or direct the spread of BYDV-GPV along the circulative transmission pathway. Thirty-three differential aphid proteins in viruliferous and nonviruliferous insects were identified using iTRAQ coupled to 2DLC-MS/MS. With the yeast two-hybrid system, 25 prey proteins were identified as interacting with the readthrough protein (RTP) and eight with the coat protein (CP), which are encoded by BYDV-GPV. Among the aphid proteins identified, most were involved in primary energy metabolism, synaptic vesicle cycle, the proteasome pathway and the cell cytoskeleton organization pathway. In a systematic comparison of the two methods, we found that the information generated by the two methods was complementary. Taken together, our findings provide useful information on the interactions between BYDV-GPV and its vector R. padi to further our understanding of the mechanisms regulating circulative transmission in aphid vectors.

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Screening for Voltage-Gated Sodium Channel Interacting Peptides

Cited by 8 publications

References 28 publications

An efficient strategy for the expression of Jingzhaotoxin-III inEscherichia coli

An efficient strategy for the expression of Jingzhaotoxin-III inEscherichia coli

Mega primer-mediated molecular cloning strategy for chimaeragenesis and long DNA fragment insertion

Integrative proteomics to understand the transmission mechanism of Barley yellow dwarf virus-GPV by its insect vector Rhopalosiphum padi

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