Cloning and expression of recombinant BmkNaTx12, a new voltage-gated sodium channel from scorpion<i> Buthus martensii </i>Karsch

XIA Yang; HU Lingling; CHEN Jiao; LU Wuguang; YAN Huaijiang; HUO Jiege; CAO Peng

doi:10.11665/j.issn.1000-5048.20170214

Journal of China Pharmaceutical University > 2017 > 48(2): 220-226. > DOI: 10.11665/j.issn.1000-5048.20170214

XIA Yang, HU Lingling, CHEN Jiao, LU Wuguang, YAN Huaijiang, HUO Jiege, CAO Peng. Cloning and expression of recombinant BmkNaTx12, a new voltage-gated sodium channel from scorpion Buthus martensii Karsch[J]. Journal of China Pharmaceutical University, 2017, 48(2): 220-226. DOI: 10.11665/j.issn.1000-5048.20170214

Citation:

PDF (2444 KB)

Cloning and expression of recombinant BmkNaTx12, a new voltage-gated sodium channel from scorpion Buthus martensii Karsch

XIA Yang^1,2,
HU Lingling^1,2,
CHEN Jiao^1,2,
LU Wuguang^1,2,
YAN Huaijiang^1,2,
HUO Jiege^1,2,
CAO Peng^1,2

More Information

Graphical Abstract

Abstract

Abstract

A cDNA library was constructed from glands of wild Buthus martensii Karsch scorpion. A new sodium channel long-chain toxin BmkNaTx12 was identified by sequence alignment, and its full-length cDNA sequence was cloned. Sequence alignment showed that the sequence of BmkNaTx12 had high structural similarity with the β-toxin Cn12 from Mexican Centruroides noxius scorpion. The predicted structure of BmkNaTx12 was obtained by homologous model construction and the highest scoring model was selected. The protein structure was found to be stable at 300 ns by molecular dynamics optimization, and fluctuations at amino acids 20, 35, and 52 are most likely due to the location of these amino acids in the loop region. Then, an expression system of recombinant BmkNaTx12-Fc fusion protein was constructed. The optimal expression time of recombinant protein was determined by Western blot and the fusion protein was successfully expressed in HEK293 cells. The purity of recombinant fusion protein which was obtained by protein A affinity chromatography was up to 95% by SDS-PAGE gel electrophoresis and HPLC. In addition, the whole-cell patch-clamp assay results suggest that 1 μmol/L BmkNaTx12-Fc can increase 20% Nav1. 7 peak current. These results laid a foundation for the further study of biological function.
- Buthus martensii Karsch,
- scorpion toxin,
- gene cloning,
- expression,
- Na⁺ channel

FullText(HTML)

References (20)

References

[1]	Cao Z,Di Z,Wu Y,et al.Overview of scorpion species from China and their toxins[J].Toxins,2014,6(3):796-815.
[2]	Shao J,Zhang R,Ge X,et al.Analgesic peptides in ButhusmartensiiKarsch:a traditional chinese animal medicine the analgesic peptides in scorpion venoms[J].Asian J Tradit Med,2007(2):45-50.
[3]	Lu W, Qiu L, Yan Z, et al. Cytotoxic T cell responses are enhanced by antigen design involving the presentation of MUC1 peptide on cholera toxin B subunit[J].Oncotarget,2015,6(33):34537-34548.
[4]	Rc LV,Possani LD.Overview of scorpion toxins specific for Na⁺ channels and related peptides:biodiversity,structure-function relationships and evolution[J].Toxicon,2005,46(8):831-844.
[5]	CamposFV,Chanda B,Beirão PS,et al.α-scorpion toxin impairs a conformational change that leads to fast inactivation of muscle sodium channels[J].J Gen Physiol,2008,132(2):251-63.
[6]	Cw GCC,Tytgat J.An overview of toxins and genes from the venom of the Asian scorpion Buthus martensi Karsch[J].Toxicon,2002,40(9):1239-1258.
[7]	Jin Y, Lee WH, Zeng L, et al. Molecular characterization of L-amino acid oxidase from king cobra venom[J].Toxicon,2007,50(4):479-489.
[8]	Zhou X,Cao P,Tian Y,et al.Expressed peptide assay for DNA detection[J].J Am ChemSoc,2010,132(12):4161-4168.
[9]	Shan LL,Gao JF,Zhang YX,et al.Proteomic characterization and comparison of venoms from two elapid snakes(Bungarusmulticinctus and Najaatra)from China[J].J Proteomics,2016,138:83-94.
[10]	Anangi R,Chen CC,Lin YW,et al.Expression in Pichia pastoris and characterization of APETx2,a specific inhibitor of acid sensing ion channel 3[J].Toxicon,2010,56(8):1388-1397.
[11]	PRF Executive Editor.Structural biology:pain-sensing TRPA1 channel resolved[J].Nature,2015,520(7548):439-441.
[12]	Possani LD,Merino E,Corona M,et al.Peptides and genes coding for scorpion toxins that affect ion-channels[J].Biochimie,2000,82(9):861-868.
[13]	Liu LH,Bosmans F,Maertens C,et al.Molecular basis of the mammalian potency of the scorpion α-like toxin,BmK M1[J].FASEB J,2005,19(6):594-596.
[14]	Ye X, Bosmans F, Li C, et al. Structural basis for the voltage-gated Na+ channel selectivity of the scorpion alpha-like toxin BmK M1[J].J Mol Biol,2005,353(4):788-803.
[15]	Xiong F,Yan B,Dai WL,et al.Analgesic effect of levo-corydalmine on neuropathic pain in rats and its mechanism[J].J China Pharm Univ(中国药科大学学报),2017,48(1):70-75.
[16]	Motin L,Durek T,Adams DJ.Modulation of human Na1.7 channel gating by synthetic α-scorpion toxin OD1 and its analogues[J].Channels,2016,10(2):139-147.
[17]	Chen L,Sun D,Wu M,et al.Cloning,expression,purification,crystallization and preliminary crystallographic analysis of Rv1698,an outer membrane channel protein from Mycobacterium tuberculosis.[J].Acta Crystallogr Sect F Struct Biol Cryst Commun,2010,66(11):1525-1527.
[18]	Maertens C,Cuypers E,Amininasab M,et al.Potent modulation of the voltage-gated sodium channel Nav1.7 by OD1,a toxin from the scorpion Odonthobuthusdoriae[J].Mol Pharmacol,2006,70(1):405-414.
[19]	O′Brien JE,Sharkey LM,Vallianatos CN,et al.Interaction of voltage-gated sodium channel Nav1.6(SCN8A)with microtubule-associated protein Map1b[J].J Biol Chem,2012,287(22):18459-18466.
[20]	Del RF,Hernández-Marín E,Pimienta G,et al.NMR solution structure of Cn12,a novel peptide from the Mexican scorpion Centruroidesnoxius with a typical beta-toxin sequence but with alpha-like physiological activity[J].Eur J Biochem,2004,271(12):2504-2516.

[1]	QIAN Xiuhui, SUN Jing, FU San, TANG Xiaoyan, XU Xianghong, ZHANG Mian. Effect of intratracheal instillation of PM_2.5suspensionon pulmonary fibrosis in mice and the intervention of neotuberostemonine[J]. Journal of China Pharmaceutical University, 2021, 52(4): 455-462. DOI: 10.11665/j.issn.1000-5048.20210408
[2]	FANG Daqing, LIU Fang, ZHANG Sijia, CAI Chaoqun, LIU Minzhuo, LIU Jinghan, YANG Chunhua, WANG Hao. One new diterpenoid alkaloid from Aconitum carmichaeli[J]. Journal of China Pharmaceutical University, 2017, 48(5): 568-571. DOI: 10.11665/j.issn.1000-5048.20170510
[3]	XIANG Juan, YU Ping, LI Mingdan, ZHANG Chaofeng, XU Xianghong, ZHANG Mian. Protective effects of stemona alkaloids on mice with bleomycin-induced pulmonary fibrosis[J]. Journal of China Pharmaceutical University, 2017, 48(1): 76-81. DOI: 10.11665/j.issn.1000-5048.20170112
[4]	YAO Juan, XIONG Fei, ZHU Zi-yi, LIU Jian-ping, GU Ning. Preparation and in vitro evaluation of vincristine-loaded superparamagnetic iron oxide nanoparticles[J]. Journal of China Pharmaceutical University, 2012, 43(3): 222-225.
[5]	Comparative pharmacokinetics of aconitine, mesaconitine and hypaconitine in rats after oral administration of four decoctions composed with Radix Aconiti Lateralis[J]. Journal of China Pharmaceutical University, 2010, 41(1): 55-59.
[10]	POLLEN MORPHOLOGY IN CHINESE SPECIES OF STEMONA[J]. Journal of China Pharmaceutical University, 1989, (6): 325-329.

Cited By

Get Citation

PDF

XML

Article views PDF downloads

Turn off MathJax

Article Contents

Abstract

References

Cloning and expression of recombinant BmkNaTx12, a new voltage-gated sodium channel from scorpion Buthus martensii Karsch

Abstract

References

Related Articles

Catalog

Cloning and expression of recombinant BmkNaTx12, a new voltage-gated sodium channel from scorpion Buthus martensii Karsch

Abstract

References

Related Articles

Catalog

Export File

Citation

Format

Content