Expression and Binding Properties of a Soluble Chimeric Protein Containing the N-Terminal Domain of the Duffy Antigen

Abstract

The blood group Duffy antigen of human erythrocytes, which exists in two allelic forms, Fya and Fyb, is a promiscuous chemokine receptor. In this report we describe the expression and purification of a chimeric protein composed of the amino-terminal extracellular domain of the Duffy antigen (aa 3–60), C-terminal intracellular fragment of glycophorin A (GPA, aa 104–131), and the hexahistydyl tag. We obtained two forms of the recombinant protein containing the Fya or Fyb epitope, denoted Fya/GPA and Fyb/GPA, respectively. These constructs were expressed in Escherichia coli as periplasmic proteins and were purified by affinity chromatography on the Ni-NTA-agarose. Both proteins bound the monoclonal antibodies recognizing the common Fy6 epitope of the Duffy antigen and an epitope of the C-terminal fragment of GPA, and only the Fya/GPA bound anti-Fya antibody. However, binding of IL-8 to the recombinant proteins was not detected, which indicated that an N-terminal domain of the Duffy antigen is not sufficient for an effective chemokine binding. The lack of the chemokine binding was not likely to be due to the lack of glycosylation of the Fy/GPA, since IL-8 was effectively bound to de-N-glycosylated erythrocytes.