title: Co-overexpression of PpPDI enhances secretion of ancrod in Pichia pastoris.
abstract: Ancrod, a serine protease purified from the venom of Agkistrodon rhodostoma, is highly specific for fibrinogen. It causes anticoagulation by defibrinogenation and has been used as a therapeutic anticoagulant for the treatment of moderate to severe forms of peripheral arterial circulatory disorders in a variety of countries. The DNA of ancrod was amplified by recursive PCR with a yeast bias codon and cloned into the pGEM-T Easy vector. In order to achieve a high level secretion and a full activity expression of ancrod in Pichia pastoris (P. pastoris), the P. pastoris protein disulfide bond isomerase (PpPDI) was co-overexpressed in the strain. The secretion characteristics of ancrod with and without PpPDI were examined. With co-overexpression of PpPDI, the production of recombinant ancrod (rAncrod) was increased to 315 mg/L in the culture medium, which is twofold higher than the control strain carrying only the ancrod gene. Through purified by Ni²⁺ affinity chromatography and phenyl Sepharose column, the purity of rAncrod was found to be as high as 95.2%. The fibrinogenolytic and zymographic activities of the rAncrod were determined and found to be similar to that of the native protein. This improved expression system can facilitate further studies and the industrial production of ancrod.
authors.affiliation: Department of Bioengineering, Zhengzhou University, Zhengzhou, Henan 450001, China. zhangst@zzu.edu.cn
authors.forename: Shou-Tao
authors.initials: ST
authors.lastname: Zhang
authors.forename: Hui-Min
authors.initials: HM
authors.lastname: Fang
authors.forename: Li
authors.initials: L
authors.lastname: Zhao
authors.forename: Qing-Nan
authors.initials: QN
authors.lastname: Tian
authors.forename: Yun-Fei
authors.initials: YF
authors.lastname: Qin
authors.forename: Ping
authors.initials: P
authors.lastname: Lu
authors.forename: San-Jun
authors.initials: SJ
authors.lastname: Chen
authors.forename: Zhen-Xia
authors.initials: ZX
authors.lastname: Bao
authors.forename: Feng
authors.initials: F
authors.lastname: Liang
mesh_terms: D017836:Agkistrodon / Q000378:metabolism*; D000721:Ancrod / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D000925:Anticoagulants / Q000378:metabolism; D015153:Blotting, Western; D002846:Chromatography, Affinity; D003435:Crotalid Venoms / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005340:Fibrinogen / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors; D010843:Pichia / Q000201:enzymology* / Q000235:genetics; D016133:Polymerase Chain Reaction; D019704:Protein Disulfide-Isomerases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D000925:Anticoagulants; D003435:Crotalid Venoms; D005656:Fungal Proteins; D011994:Recombinant Proteins; D005340:Fibrinogen; D000721:Ancrod; D012697:Serine Endopeptidases; D019704:Protein Disulfide-Isomerases