Soluble expression and one-step purification of a neurotoxin Huwentoxin-I in Escherichia coli

Protein Expr Purif. 2009 Jun;65(2):154-9. doi: 10.1016/j.pep.2009.02.002. Epub 2009 Feb 13.

Abstract

Huwentoxin-I (HWTX-I) is a small 33-amino acid neurotoxin from the venom of the Chinese bird spider Ornithoctonus huwena. HWTX-I selectively blocks N-type voltage-sensitive calcium channels (N-VSCCs) and has great potential for clinical application as a novel analgesic without inducing drug tolerance. However, there are still many unsolved issues for this peptide, such as its clinical efficacy in analgesia, anesthesia, and even its potential role in drug rehabilitation. Therefore, large amounts of active recombinant HWTX-I are urgently needed. In this report, we describe a novel and efficient way to produce large amounts of the valuable form in Escherichia coli. HWTX-I was expressed in soluble form as an N-terminal intein fusion product. After affinity purification, a pH shift-induced self-cleavage of the intein released HWTX-I, resulting in a single-column purification of the target protein. The whole-cell patch clamp assay showed that purified HWTX-I has activity similar to another commercialized N-VSCC blocker omega-conotoxin MVIIA. Production of HWTX-I by this method has the major advantages of high efficiency and low cost.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Calcium Channels, N-Type / drug effects
  • Calcium Channels, N-Type / metabolism
  • Cerebral Cortex / drug effects
  • Cerebral Cortex / metabolism
  • China
  • Drug Tolerance
  • Escherichia coli / genetics*
  • Escherichia coli / metabolism
  • Neurons / drug effects
  • Neurons / metabolism
  • Neurotoxins / biosynthesis*
  • Neurotoxins / genetics
  • Neurotoxins / isolation & purification*
  • Neurotoxins / pharmacology
  • Rats
  • Rats, Sprague-Dawley
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / pharmacology
  • Reptilian Proteins / biosynthesis*
  • Reptilian Proteins / genetics
  • Reptilian Proteins / isolation & purification*
  • Reptilian Proteins / pharmacology
  • Solubility
  • Spider Venoms / biosynthesis*
  • Spider Venoms / genetics
  • Spider Venoms / isolation & purification*
  • Spider Venoms / pharmacology
  • Spiders / chemistry*
  • Spiders / genetics

Substances

  • Calcium Channels, N-Type
  • Neurotoxins
  • Recombinant Proteins
  • Reptilian Proteins
  • Spider Venoms
  • huwentoxin I