Production and purification of human indoleamine 2,3-dioxygenase (HuIDO) protein in a baculovirus expression system and production and characterization of egg yolk antibody against the purified HuIDO.
The human indoleamine 2,3-dioxygenase (HuIDO) baculoviral construct, for expression of HuIDO protein with a hexa-histidine and FLAG (DYKDDDDK) tag, was produced using the BacPAK Baculovirus Expression System. HuIDO baculovirus was used to infect Sf21 insect cells to produce functionally active protein in large amounts. Conditions for protein purification by metal affinity chromatography were determined and optimized. Addition of haemin ensured optimal activity of the purified heme-containing oxygenase. The soluble purified protein was used to immunize a chicken to produce large quantities of polyclonal IgY against HuIDO. The anti-HuIDO IgY antibody specifically detected HuIDO produced by a range of cell types including transfectants and native HuIDO expression induced in IFN-gamma-stimulated cells. The antibody detected HuIDO in cell lysates by western blotting and in the cytoplasm of cells by microscopy. The antibody was unable to block the function of the enzyme, indicating that this antibody binds outside the active site of HuIDO.
Webster
Nicole L
NL
Department of Microbiology and Immunology, The University of Melbourne, Melbourne, Australia.
Wee
Janet
J
Uren
Sandra J
SJ
Boyle
William
W
Sandrin
Mauro S
MS
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002642:Chick Embryo; D003001:Cloning, Molecular; D004530:Egg Yolk / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors / Q000138:chemical synthesis; D006427:Hemin / Q000494:pharmacology; D006801:Humans; D007136:Immunoglobulins / Q000096:biosynthesis / Q000737:chemistry*; D014366:Tryptophan Oxygenase / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism
C016512:IgY; D007136:Immunoglobulins; D006427:Hemin; D014366:Tryptophan Oxygenase