title: In vitro refolding and functional analysis of polyhistidine-tagged Buthus martensii Karsch antitumor-analgesic peptide produced in Escherichia coli.
abstract: OBJECTIVE
  To identify an efficient in vitro refolding method to generate highly active His6-tagged scorpion toxin antitumor-analgesic peptide (AGAP) isolated from Escherichia coli inclusion bodies.
  
  
  RESULTS
  N- and C-Terminal His6-tagged recombinant (r) AGAP (NHis6-rAGAP and CHis6-rAGAP, respectively) were expressed in E. coli; the purification and refolding conditions were optimized. CHis6-rAGAP, but not NHis6-rAGAP, exhibited significant in vitro antihepatoma activity that was much greater than that of rAGAP produced using SUMO fusion technology (IC50, 0.4 ± 0.08 vs. 1.8 ± 0.3 μM). CHis6-rAGAP also showed significant inhibition of tumor growth in a mouse xenograft model of human hepatoma and inhibition of neuronal excitability, demonstrated by blockage of voltage-sensitive tetrodotoxin-resistant (TTX-R) sodium currents in acute isolated dorsal root ganglion neurons.
  
  
  CONCLUSIONS
  This refolding protocol optimized for C-terminal His6-tagged scorpion rAGAP is potentially applicable to similar long-chain and cysteine-rich toxins.
authors: Changhai Hospital of Traditional Chinese Medicine, Second Military Medical University, Shanghai, 200433, China.|, 455 Military Hospital, Shanghai, 200052, China.
authors: Qingxin
authors: Q
authors: Cao
authors: Laboratory of Cellular and Molecular Biology, Jiangsu Province Institute of Traditional Chinese Medicine, 100#, Shizi Street, Hongshang Road, Nanjing, 210028, Jiangsu, China.
authors: Wuguang
authors: W
authors: Lu
authors: Laboratory of Cellular and Molecular Biology, Jiangsu Province Institute of Traditional Chinese Medicine, 100#, Shizi Street, Hongshang Road, Nanjing, 210028, Jiangsu, China.
authors: Xueting
authors: X
authors: Cai
authors: Laboratory of Cellular and Molecular Biology, Jiangsu Province Institute of Traditional Chinese Medicine, 100#, Shizi Street, Hongshang Road, Nanjing, 210028, Jiangsu, China.
authors: Chunping
authors: C
authors: Hu
authors: Changhai Hospital of Traditional Chinese Medicine, Second Military Medical University, Shanghai, 200433, China.
authors: Chen
authors: C
authors: Wang
authors: Laboratory of Cellular and Molecular Biology, Jiangsu Province Institute of Traditional Chinese Medicine, 100#, Shizi Street, Hongshang Road, Nanjing, 210028, Jiangsu, China.
authors: Juan
authors: J
authors: Ye
authors: Laboratory of Cellular and Molecular Biology, Jiangsu Province Institute of Traditional Chinese Medicine, 100#, Shizi Street, Hongshang Road, Nanjing, 210028, Jiangsu, China.
authors: Huaijiang
authors: H
authors: Yan
authors: Institute of Medical Science and Department of Laboratory for Molecular Structure and Function of Drug-Target Proteins, Shanghai Jiaotong University School of Medicine, Shanghai, 200025, China.
authors: Yang
authors: Y
authors: Yang
authors: Laboratory of Cellular and Molecular Biology, Jiangsu Province Institute of Traditional Chinese Medicine, 100#, Shizi Street, Hongshang Road, Nanjing, 210028, Jiangsu, China.
authors: Zhigang
authors: Z
authors: Wang
authors: Changhai Hospital of Traditional Chinese Medicine, Second Military Medical University, Shanghai, 200433, China.|Laboratory of Cellular and Molecular Biology, Jiangsu Province Institute of Traditional Chinese Medicine, 100#, Shizi Street, Hongshang Road, Nanjing, 210028, Jiangsu, China.|Institute of Medical Science and Department of Laboratory for Molecular Structure and Function of Drug-Target Proteins, Shanghai Jiaotong University School of Medicine, Shanghai, 200025, China.|, 455 Military Hospital, Shanghai, 200052, China.
authors: Jiege
authors: J
authors: Huo
authors: Institute of Medical Science and Department of Laboratory for Molecular Structure and Function of Drug-Target Proteins, Shanghai Jiaotong University School of Medicine, Shanghai, 200025, China.
authors: Yan
authors: Y
authors: Liu
authors: Institute of Medical Science and Department of Laboratory for Molecular Structure and Function of Drug-Target Proteins, Shanghai Jiaotong University School of Medicine, Shanghai, 200025, China.
authors: Ye
authors: Y
authors: Yu
authors: Changhai Hospital of Traditional Chinese Medicine, Second Military Medical University, Shanghai, 200433, China. lingchangquan@hotmail.com.
authors: Changquan
authors: C
authors: Ling
authors: Laboratory of Cellular and Molecular Biology, Jiangsu Province Institute of Traditional Chinese Medicine, 100#, Shizi Street, Hongshang Road, Nanjing, 210028, Jiangsu, China. pcao79@yahoo.com.
authors: Peng
authors: P
authors: Cao
mesh_terms: D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy; D004195:Disease Models, Animal; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D064593:Heterografts; D006639:Histidine / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000627:therapeutic use; D006801:Humans; D051379:Mice; D009474:Neurons / Q000187:drug effects; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000627:therapeutic use; D012604:Scorpion Venoms / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000627:therapeutic use; D016896:Treatment Outcome; D061566:Voltage-Gated Sodium Channels / Q000187:drug effects
chemical_list: D011993:Recombinant Fusion Proteins; D012604:Scorpion Venoms; D061566:Voltage-Gated Sodium Channels; C477804:antitumor-analgesic peptide, Buthus martensii; C033223:polyhistidine; D006639:Histidine
keywords: Anti-tumor peptide; His6-tagged rAGAP; Inclusion body; Molecular dynamics simulation; Scorpion toxin; Tetrodotoxin-resistant sodium currents