Eukaryotic expression and purification of anti-epilepsy peptide of Buthus martensii Karsch and its protein interactions.
The Asian scorpion Buthus martensil Karsch is important in the Chinese traditional medicine where it is used for the treatment of some nervous system diseases. The anti-epilepsy peptide (AEP) is a 61-amino-acid polypeptide extracted from the venom of B. martensil Karsch. Research has confirmed that it has anti-epileptic effects on the rat model of epilepsy. In this experiment, a cDNA library of AEP from the venom of B. martensil Karsch was constructed using RT-PCR; the primer was designed and used for the amplification. An expression vector of AEP was constructed using Pichia pastoris. Vector expression was induced, and protein purification was then performed. Bolting of the interaction molecule of AEP was by His pull down. Experimental results indicate high AEP expression, and the obtained protein was purified and compared with the control group. Four conspicuous protein bands were observed, and mass chromatographic analysis indicated that the four proteins were synaptosomal-associated protein of 25 kDa (SNAP-25), glial fibrillary acidic protein (GFAP), Glutamic acid decarboxylase (GAD) and N-methyl-D: -aspartate (NMDA). Further, the four protein bands were verified by mammalian two-hybrid experiments and co-immunoprecipitation. AEP was found to interact with SNAP2 and NMDA. This provides experimental evidence for the mechanism of AEP's anti-epileptic action and for the manufacture of a novel type anti-epileptic drug.
Wang
Zongren
Z
Department of Traditional Chinese Medicine, Xijing Hospital, The Forth Military Medical University, Xi'an, Shaanxi, 710032, People's Republic of China. zongren@fmmu.edu.cn
Wang
Wen
W
Shao
Zhongjun
Z
Gao
Bifeng
B
Li
Junchang
J
Ma
Jing
J
Li
Jinghua
J
Che
Honglei
H
Zhang
Wei
W
D000818:Animals; D003001:Cloning, Molecular / Q000379:methods*; D004827:Epilepsy / Q000188:drug therapy*; D005822:Genetic Vectors; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D005968:Glutamate Decarboxylase / Q000378:metabolism; D006624:Hippocampus / Q000737:chemistry / Q000166:cytology; D016202:N-Methylaspartate / Q000378:metabolism; D010843:Pichia / Q000235:genetics; D011485:Protein Binding; D025941:Protein Interaction Mapping; D051381:Rats; D017207:Rats, Sprague-Dawley; D012604:Scorpion Venoms / Q000235:genetics / Q000494:pharmacology*; D050825:Synaptosomal-Associated Protein 25 / Q000378:metabolism
D005904:Glial Fibrillary Acidic Protein; D012604:Scorpion Venoms; D050825:Synaptosomal-Associated Protein 25; C058792:anti-epilepsy peptide, Buthrus martensii; D016202:N-Methylaspartate; D005968:Glutamate Decarboxylase