title: Optimized production of scygonadin in Pichia pastoris and analysis of its antimicrobial and antiviral activities.
abstract: The crab antimicrobial peptide scygonadin is confirmed to have antimicrobial activity against bacteria and it is probably associated with the reproductive immunity in Scylla paramamosain. To obtain large quantity of scygonadin for further biological assays, a 306 bp cDNA sequence encoding the mature peptide of scygonadin was cloned into a secretion vector of pPIC9K, and a high-level of the recombinant scygonadin was achieved in Pichia pastoris. The optimal expression condition was determined as incubation with 0.5% methanol for 48 h at 28 °C under pH 6.0, and a total of 70 mg scygonadin was expressed in 1L culture medium. The recombinant product was purified and 97% pure scygonadin was obtained using immobilized metal affinity chromatography with a yield of 46 mg/L. The recombinant scygonadin was confirmed using SDS-PAGE analysis and MS-fingerprinting. P. pastoris-derived scygonadin exhibited relatively higher antimicrobial activities against bacteria than Escherichia coli-derived scygonadin. The antimicrobial activity of the recombinant scygonadin against pathogenic Aeromonas hydrophila showed salt resistant and the killing kinetics of A. hydrophila was time dependent. Besides, the antiviral assay demonstrated that scygonadin could interfere with white spot syndrome virus (WSSV) replication in vitro-cultured crayfish haematopoietic (Hpt) cells. Taken together, this is the first report on the heterologous expression of scygonadin in P. pastoris, and P. pastoris is an effective expression system for producing large quantities of biological active scygonadin for both research and agricultural application.
authors: State Key Laboratory of Marine Environmental Science, College of Oceanography and Environmental Science, Xiamen University, Xiamen, Fujian 361005, PR China.
authors: Hui
authors: H
authors: Peng
authors: Hai-Peng
authors: HP
authors: Liu
authors: Bei
authors: B
authors: Chen
authors: Hua
authors: H
authors: Hao
authors: Ke-Jian
authors: KJ
authors: Wang
mesh_terms: D000818:Animals; D000890:Anti-Infective Agents / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D000998:Antiviral Agents / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D017756:Aquaculture; D060829:Arthropod Proteins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification / Q000494:pharmacology; D003400:Astacoidea / Q000737:chemistry / Q000276:immunology; D001419:Bacteria / Q000187:drug effects; D003386:Brachyura / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D002478:Cells, Cultured; D015870:Gene Expression; D005822:Genetic Vectors; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D014780:Viruses / Q000187:drug effects
chemical_list: D000890:Anti-Infective Agents; D000998:Antiviral Agents; D060829:Arthropod Proteins; D011994:Recombinant Proteins