title: Expression of serine proteinase P186 of Arthrobotrys oligospora and analysis of its nematode-degrading activity.
abstract: The nematode-trapping fungi possess a unique capability of predating and invading nematodes. As a representative nematode-trapping fungus, Arthrobotrys oligospora has been widely used to study the interactions between nematode-trapping fungi and their hosts. Serine proteinase is one of the important virulence factors during process of invasion of the nematode-trapping fungi into nematodes. In this study, using reverse transcription polymerase chain reaction, we amplified the gene sequence of serine proteinase 186 from A. oligospora, cloned it into pPIC9K vector and expressed it in the yeast Pichia pastoris. The expressed recombinant serine proteinase186 (reP186) was purified via Ni-affinity chromatography. The in vitro nematode-degrading activity of reP186 was analyzed. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis revealed that reP186 with molecular weight of 33 kDa was successfully obtained. ReP186 was capable of degrading a series of protein substrates including casein, gelatin, bovine serum albumin, denatured collagen and nematode cortical layer. The reP186 exhibited the maximal activity at pH 8.0 and 55 °C and was highly sensitive to the inhibitor, phenylmethanesulfonylfluoride. Treatment of Caenorhabditis elegans and Haemonchus contortus with reP186 for 12, 24 and 36 h, respectively, resulted in 62, 88 and 100 % of killing rates for C. elegans, and 52, 65 and 84 % of killing rates for H. contortus, respectively, indicating a relatively strong nematode-degrading bioactivity of reP186.
authors.affiliation: Key Laboratory of Control and Prevention of Animal Disease of Xinjiang Production & Construction Corps, College of Animal Science and Technology, Shihezi University, North Street No.4, Shihezi, 832003, Xinjiang, China.
authors.forename: Hailong
authors.initials: H
authors.lastname: Zhao
authors.affiliation: Key Laboratory of Control and Prevention of Animal Disease of Xinjiang Production & Construction Corps, College of Animal Science and Technology, Shihezi University, North Street No.4, Shihezi, 832003, Xinjiang, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Qiao
authors.affiliation: Key Laboratory of Control and Prevention of Animal Disease of Xinjiang Production & Construction Corps, College of Animal Science and Technology, Shihezi University, North Street No.4, Shihezi, 832003, Xinjiang, China. xjmqlqj@sina.com.
authors.forename: Qingling
authors.initials: Q
authors.lastname: Meng
authors.affiliation: Key Laboratory of Control and Prevention of Animal Disease of Xinjiang Production & Construction Corps, College of Animal Science and Technology, Shihezi University, North Street No.4, Shihezi, 832003, Xinjiang, China.
authors.forename: Shasha
authors.initials: S
authors.lastname: Gong
authors.affiliation: Key Laboratory of Control and Prevention of Animal Disease of Xinjiang Production & Construction Corps, College of Animal Science and Technology, Shihezi University, North Street No.4, Shihezi, 832003, Xinjiang, China.
authors.forename: Cheng
authors.initials: C
authors.lastname: Chen
authors.affiliation: Key Laboratory of Control and Prevention of Animal Disease of Xinjiang Production & Construction Corps, College of Animal Science and Technology, Shihezi University, North Street No.4, Shihezi, 832003, Xinjiang, China.
authors.forename: Tianli
authors.initials: T
authors.lastname: Liu
authors.affiliation: Key Laboratory of Control and Prevention of Animal Disease of Xinjiang Production & Construction Corps, College of Animal Science and Technology, Shihezi University, North Street No.4, Shihezi, 832003, Xinjiang, China.
authors.forename: Lulu
authors.initials: L
authors.lastname: Tian
authors.affiliation: State Key Lab of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, 730046, Gansu, China.
authors.forename: Xuepeng
authors.initials: X
authors.lastname: Cai
authors.affiliation: State Key Lab of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, 730046, Gansu, China.
authors.forename: Jianxun
authors.initials: J
authors.lastname: Luo
authors.affiliation: Key Laboratory of Control and Prevention of Animal Disease of Xinjiang Production & Construction Corps, College of Animal Science and Technology, Shihezi University, North Street No.4, Shihezi, 832003, Xinjiang, China.
authors.forename: Chuangfu
authors.initials: C
authors.lastname: Chen
mesh_terms: D000818:Animals; D001203:Ascomycota / Q000201:enzymology*; D015153:Blotting, Western; D017173:Caenorhabditis elegans / Q000187:drug effects* / Q000502:physiology; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006189:Haemonchus / Q000187:drug effects* / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011480:Protease Inhibitors / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D057057:Serine Proteases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016019:Survival Analysis; D013696:Temperature; D014105:Tosyl Compounds / Q000378:metabolism
chemical_list: D011480:Protease Inhibitors; D011994:Recombinant Proteins; D014105:Tosyl Compounds; C003614:4-toluenesulfonyl fluoride; D057057:Serine Proteases
keywords: Arthrobotrys oligospora; Eukaryotic expression; Nematode-degrading activity; Pichia pastoris; Serine proteinase P186