[Expression and purification of recombinant huwentoxin-I in Pichia pastoris].
HWTX-I is a peptide neurotoxin purified from the crude venom of the Chinese bird Spider Selenocosmia Huwena, which has analyesic activity. rHWTX-I expressed by P. pastoris and secreted to culture supernatant was first precipitated by (NH4)2SO4, then it was isolated and desalted by ultrofiltration following by ion exchange chromatography of CM column, after reverse phase HPLC of C18 column and vacuum drying, the pure HWTX-I protein was obtained which was proved to be recombinant HWTX-I by Tricine SDS-PAGE, MALDI-TOF mass spectrometry, amino acid composition analysis, the N-terminal amino acid sequence and its biological activity. The final yield of the purified HWTX-I was about 80 mg/L accounting for 23.6% of its total secretory proteins.
Nie
Dong-Song
DS
College of Life Science, Hunan Normal University, Changsha 410081, China.
Li
Min
M
Xu
Hui-Ming
HM
He
Ning-Jia
NJ
Liang
Song-Ping
SP
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D003470:Culture Media; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D009469:Neuromuscular Junction / Q000187:drug effects; D009498:Neurotoxins / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D010843:Pichia; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D030162:Reptilian Proteins; D012671:Seminiferous Tubules / Q000187:drug effects; D020539:Sequence Analysis, Protein; D013111:Spider Venoms / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D013112:Spiders; D009435:Synaptic Transmission / Q000187:drug effects; D013997:Time Factors
D003470:Culture Media; D009498:Neurotoxins; D011993:Recombinant Fusion Proteins; D030162:Reptilian Proteins; D013111:Spider Venoms; C083624:huwentoxin I