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.
Keywords:
Bioactivity; LECT2; Oncorhynchus mykiss; Pichia pastoris; Purification.
MeSH terms
-
Animals
-
Blotting, Western
-
Cathepsin D / genetics
-
Chromatography, Gel
-
Chromatography, Ion Exchange
-
Cytotoxicity, Immunologic / drug effects
-
Escherichia coli / growth & development
-
Escherichia coli / immunology
-
Fish Proteins / genetics
-
Fish Proteins / metabolism*
-
Gene Expression / drug effects
-
Head Kidney / cytology
-
Intercellular Signaling Peptides and Proteins / genetics
-
Intercellular Signaling Peptides and Proteins / metabolism*
-
Intercellular Signaling Peptides and Proteins / pharmacology
-
Macrophage Activation / drug effects
-
Macrophage Activation / immunology
-
Macrophages / drug effects
-
Macrophages / immunology
-
Macrophages / metabolism
-
Oncorhynchus mykiss / genetics
-
Oncorhynchus mykiss / metabolism*
-
Pichia / genetics
-
Receptors, CCR / genetics
-
Recombinant Proteins / isolation & purification
-
Recombinant Proteins / metabolism*
-
Recombinant Proteins / pharmacology
-
Respiratory Burst / drug effects
-
Reverse Transcriptase Polymerase Chain Reaction
Substances
-
CC chemokine receptor 9
-
Fish Proteins
-
Intercellular Signaling Peptides and Proteins
-
Receptors, CCR
-
Recombinant Proteins
-
Cathepsin D