Expression and purification of recombinant human N-formyl-L-leucyl-L-phenylalanine (FMLP) receptor: generation of polyclonal antibody against FMLP receptor.
The recombinant formyl peptide receptor has been successfully expressed and purified, utilizing an Escherichia coli expression system. Purification of formyl peptide receptor was performed using gel filtration chromatography and affinity chromatography, and the purified protein migrated at an apparent molecular mass of 36,000 Da. The purified recombinant receptor retained functional activity as determined by a ligand binding assay. The yield of the recombinant purified receptor was approximately 1 mg/2 L of culture, and the binding activity was determined to be approximately 8 nM, which suggests the conclusion that glycosylation does not affect significantly ligand binding of the N-formyl-L-leucyl-L-phenylalanine (FMLP) receptor molecule. The recombinant receptor protein yield was found to be significantly higher than that obtained from neutrophils. The purified recombinant receptor was then utilized to generate antibody against the same. The reaction of the antibody against recombinant formylpeptide receptor and against native formylpeptide receptor on neutrophils was confirmed by western blot analysis and flow cytometric analysis, respectively. The antibody was also used successfully to detect recombinant formylpeptide receptor expression on transfected 293 cells. These results describe for the first time the expression, purification, and characterization of recombinant FMLP receptor with ligand binding activity and the generation of polyclonal antibody against the same. This work also provides a foundation for future biophysical studies of the FMLP receptor molecule, which have not been possible until now.
Lala
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Department of Oral Biology, State University of New York at Buffalo, 14214, U.S.A.
Sojar
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HT
De Nardin
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D000906:Antibodies / Q000032:analysis*; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D005434:Flow Cytometry; D005822:Genetic Vectors; D006801:Humans; D009504:Neutrophils / Q000378:metabolism; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D018000:Receptors, Peptide / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis; D014162:Transfection
D000906:Antibodies; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D018000:Receptors, Peptide; D011994:Recombinant Proteins