title: [Yeast-based production, purification and bioactivity assay of rainbow trout LECT2].
abstract: Leukocyte cell-derived chemotaxin 2 (LECT2) is a secretory cytokine that functions in many physiological and pathological processes. We used a Pichia pastoris expression system for the recombinant expression of rainbow trout LECT2. The recombinant LECT2 was purified by UNOsphere S Cation exchange and size-exclusion chromatography columns. The obtained target protein was highly pure (>96% homogeneity) and the yield was >120 mg/L of yeast cultures. An in vitro chamber assay revealed that recombinant LECT2 could induce chemotactic responses in rainbow trout head kidney-derived macrophages. Recombinant LECT2 not only enhanced macrophage respiratory burst activity and bactericidal activity, but also changed macrophage gene expression. In summary, we established a rapid and efficient method to prepare active recombinant rainbow trout LECT2 using a yeast expression system and column chromatography. Bioactive recombinant LECT2 is essential for studies on protein functions.
authors: Key laboratory of Applied Marine Biotechnology, Ministry of Education, Ningbo University, Ningbo 315211, China.
authors: Yu-Hong
authors: YH
authors: Shi
authors: Rui-Cheng
authors: RC
authors: Zhang
authors: Dan-Yang
authors: DY
authors: Yang
authors: Xin-Jiang
authors: XJ
authors: Lu
authors: Jiong
authors: J
authors: Chen
authors: Min-Yun
authors: MY
authors: Li
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002402:Cathepsin D / Q000235:genetics; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003602:Cytotoxicity, Immunologic / Q000187:drug effects; D004926:Escherichia coli / Q000254:growth & development / Q000276:immunology; D029941:Fish Proteins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects; D060226:Head Kidney / Q000166:cytology; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D008262:Macrophage Activation / Q000187:drug effects / Q000276:immunology; D008264:Macrophages / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D017686:Oncorhynchus mykiss / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000235:genetics; D054388:Receptors, CCR / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D016897:Respiratory Burst / Q000187:drug effects; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: C119309:CC chemokine receptor 9; D029941:Fish Proteins; D036341:Intercellular Signaling Peptides and Proteins; D054388:Receptors, CCR; D011994:Recombinant Proteins; D002402:Cathepsin D
keywords: Bioactivity; LECT2; Oncorhynchus mykiss; Pichia pastoris; Purification