Affinity purification of serine proteinase from Deinagkistrodon acutus venom.
An affinity protocol was developed for the preparation of the main serine proteinase from Deinagkistrodon acutus venom on industrial scales. As affinity ligand, l-arginine was composed to medium and its structure was confirmed by ESI-MS analysis. The purification process consisted of one major affinity chromatography step to remove more than 95% of other proteins, and a polishing step of DEAE ion-exchange chromatography for removal of minor contaminants. The serine proteinase was 100% pure analyzed on HPLC Vydac C4 column, 99.4% on TSK G3000SW column, and 97.7% with SDS-PAGE analysis. The yield of the main serine proteinase was 3.6% of crude venom protein, the recoveries of typical fibrinogen (Fg) clotting activity and arginine esterase activity of serine proteinase were 82.2% and 84%, higher than those of other reported traditional protocols, the proteinase also showed arginine amidase activity. Reducing SDS-PAGE analysis showed that the arginine esterase was a single polypeptide with the mass of approximately 40 kDa, while MALDI-TOF-TOF-MS analysis showed that the purified proteinase should be a approximately 34 kDa glycoprotein. The desorption constant Kd and the theoretical maximum absorption Qmax on the affinity medium were 9.93 x 10(-5) and 38.1mg/g medium in absorption analysis.
Xin
Yu
Y
Key Laboratory of Microbial Metabolism of Ministry of Education, Shanghai Jiao Tong University, College of Life Science and Biotechnology, 800 Dongchuan Road, Shanghai 200240, China.
Dong
Dexian
D
Wang
Ting
T
Li
Rongxiu
R
D000327:Adsorption; D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D003435:Crotalid Venoms / Q000201:enzymology*; D008024:Ligands; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification*; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D017819:Viperidae / Q000378:metabolism*
D003435:Crotalid Venoms; D008024:Ligands; D012697:Serine Endopeptidases