Expression and binding properties of a soluble chimeric protein containingthe N-terminal domain of the Duffy antigen

The blood group Huffy antigen of human erythrocytes, which exists in two al lelic forms, Fy(a) and Fy(b), is a promiscuous chemokine receptor. In this report we describe the expression and purification of a chimeric protein co mposed of the amino-terminal extracellular domain of the Huffy 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 protei n containing the Fy(a) or Fy(b) epitope, denoted Fy(a)/GPA and Fy(b)/GPA, r espectively. These constructs were expressed in Escherichia coli as peripla smic proteins and were purified by affinity chromatography on the Ni-NTA-ag arose. Both proteins bound the monoclonal antibodies recognizing the common Fy6 epitope of the Huffy antigen and an epitope of the C-terminal fragment of GPA, and only the Fy(a)/GPA bound anti-Fy(a) antibody. However, binding of IL-8 to the recombinant proteins was not detected, which indicated that an N-terminal domain of the Huffy antigen is not sufficient for an effecti ve 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 effective ly bound to de-N-glycosylated erythrocytes. (C) 2000 Academic Press.