Direct expression of the extracellular portion of human FcepsilonRIalpha chain as inclusion bodies in Escherichia coli.
The extracellular portion of the alpha chain of the human high-affinity IgE receptor (FcepsilonRIalpha) was expressed as inclusion bodies in Escherichia coli. In immunoblot analysis, two bands were reactive to human IgE and mouse anti-human FcepsilonRIalpha monoclonal antibodies. N-terminal sequencing showed that the two bands were equivalent to the soluble FcepsilonRIalpha with a methionine residue at the N-terminus (Met-1-172) and 23-172, in which the N-terminal 22 residues of the soluble FcepsilonRIalpha have been removed, possibly by degradation in E. coli cells. IgE-binding to CHO cells expressing FcepsilonRI was inhibited by the addition of the recombinant products prepared by the refolding procedure from inclusion bodies. The system for the expression of soluble human FcepsilonRIalpha in E. coli presented in this study and its further improvement would be useful for the production of the protein as a potent therapeutic and for analysis of the IgE-FcepsilonRIalpha interaction.
Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan. toshiro.takai@asahibeer.co.jp
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D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D015151:Immunoblotting; D007073:Immunoglobulin E / Q000378:metabolism; D002479:Inclusion Bodies / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D017455:Receptors, IgE / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D010446:Peptide Fragments; D017455:Receptors, IgE; D011994:Recombinant Proteins; D007073:Immunoglobulin E