Expression and binding properties of a soluble chimeric protein containing the N-terminal domain of the Duffy antigen.
The blood group Duffy antigen of human erythrocytes, which exists in two allelic forms, Fy(a) and Fy(b), 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 Fy(a) or Fy(b) epitope, denoted Fy(a)/GPA and Fy(b)/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 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 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.
Waśniowska
K
K
Department of Immunochemistry, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Rudolfa Weigla 12, Wroclaw, 53-114, Poland. wasniows@immuno.iitd.pan.wroc.pl
Czerwiński
M
M
Jachymek
W
W
Lisowska
E
E
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D004375:Duffy Blood-Group System / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D066298:In Vitro Techniques; D016209:Interleukin-8 / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012995:Solubility
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D004274:DNA, Recombinant; D004375:Duffy Blood-Group System; D016209:Interleukin-8; D011993:Recombinant Fusion Proteins