PMID: 9179299Jun 1, 1997Paper

In vitro folding and functional analysis of an anti-insect selective scorpion depressant neurotoxin produced in Escherichia coli

Protein Expression and Purification
Michael TurkovMichael Gurevitz

Abstract

The selective toxicity of depressant scorpion neurotoxins to insects is useful in studying insect sodium channel gating and has an applied potential. In order to establish a genetic system enabling a structure-activity approach, the functional expression of such polypeptides is required. By engineering the cDNA encoding the depressant scorpion neurotoxin, LahIT2, behind the T7 promoter, large amounts of recombinant insoluble and nonactive toxin were obtained in Escherichia coli. Following denaturation and reduction, the recombinant protein, constructed with an additional N-terminal methionine residue, was subjected to renaturation. Optimal conditions for reconstitution of a functional toxin, having a dominant fold over many other possible isoforms, were established. The recombinant active toxin was purified by RP-HPLC and characterized. Toxicity (ED50) to insects, binding affinity (IC50) to an insect receptor site, and electrophysiological effect on an insect axonal preparation were found to be similar to those of the native toxin. Substitution of the C-terminal glycine by a Gly-Lys-Lys triplet did not abolish folding but affected toxicity (3.5-fold decrease) of LqhIT2. Apparently, this efficient bacterial expression system (50...Continue Reading

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Citations

Dec 19, 2009·Molecular Biology and Evolution·Hagar WeinbergerMichael Gurevitz
Jul 27, 2005·Molecular Plant-microbe Interactions : MPMI·Gal NissanIsaac Barash
Jan 29, 2003·Proceedings of the National Academy of Sciences of the United States of America·Bora InceogluBruce D Hammock
Mar 12, 2010·Toxicon : Official Journal of the International Society on Toxinology·Yuzhe YuanShunyi Zhu
Feb 24, 2009·Toxicon : Official Journal of the International Society on Toxinology·Blanca I García-GómezLourival D Possani
Jun 5, 2007·Biochimica Et Biophysica Acta·Georgina EstradaGerardo Corzo
Jan 16, 2007·Comparative Biochemistry and Physiology. Toxicology & Pharmacology : CBP·M E De LimaM Pelhate
Jun 6, 2003·European Journal of Biochemistry·Dalia GordonMichael Gurevitz
Dec 16, 2006·Protein Expression and Purification·Yuzhe YuanShunyi Zhu
Nov 9, 2005·Toxicon : Official Journal of the International Society on Toxinology·Ricardo C Rodríguez de la Vega, Lourival D Possani
Jun 5, 2012·Toxicon : Official Journal of the International Society on Toxinology·Shih Chieh ChangRaymond S Norton
Oct 8, 2011·Toxicon : Official Journal of the International Society on Toxinology·V Quintero-HernándezL D Possani
Feb 22, 2008·Protein Expression and Purification·Cheng-Gang XuAi-Hua Liang
Feb 22, 2012·Protein Expression and Purification·Hui DaiWenxin Li
Oct 23, 2012·Toxicon : Official Journal of the International Society on Toxinology·M A TekookU Kirchhefer
Jun 7, 2016·Toxicon : Official Journal of the International Society on Toxinology·Georgina EstradaGerardo Corzo
Sep 28, 2016·Protein Engineering, Design & Selection : PEDS·Shangfei ZhangShunyi Zhu
Aug 1, 2006·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·Lior CohenDalia Gordon
Sep 1, 2011·History of Psychiatry·Greg Eghigian
May 10, 2001·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·M GurevitzO Froy
Feb 20, 1999·The Journal of Biological Chemistry·O FroyM Gurevitz
Jun 19, 2015·The Journal of Venomous Animals and Toxins Including Tropical Diseases·Herlinda ClementGerardo Corzo
Jun 3, 2018·Applied Microbiology and Biotechnology·Fernanda Gobbi AmorimEliane Candiani Arantes
Oct 29, 1998·Toxicon : Official Journal of the International Society on Toxinology·M GurevitzN Chejanovsky
Jul 2, 2021·The Biochemical Journal·Boris S ZhorovKe Dong
Jan 2, 2007·Toxicon : Official Journal of the International Society on Toxinology·Michael GurevitzDalia Gordon

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