title: Affinity purification of serine proteinase from Deinagkistrodon acutus venom.
abstract: 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.
authors: 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.
authors: Yu
authors: Y
authors: Xin
authors: Dexian
authors: D
authors: Dong
authors: Ting
authors: T
authors: Wang
authors: Rongxiu
authors: R
authors: Li
mesh_terms: 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*
chemical_list: D003435:Crotalid Venoms; D008024:Ligands; D012697:Serine Endopeptidases