Biochemical, biophysical, and functional characterization of bacterially expressed and refolded receptor binding domain of Plasmodium vivax duffy-binding protein.
Invasion of erythrocytes by malaria parasites is mediated by specific molecular interactions. Plasmodium vivax is completely dependent on interaction with the Duffy blood group antigen to invade human erythrocytes. The P. vivax Duffy-binding protein, which binds the Duffy antigen during invasion, belongs to a family of erythrocyte-binding proteins that also includes Plasmodium falciparum sialic acid binding protein and Plasmodium knowlesi Duffy binding protein. The receptor binding domains of these proteins lie in a conserved, N-terminal, cysteine-rich region, region II, found in each of these proteins. Here, we have expressed P. vivax region II (PvRII), the P. vivax Duffy binding domain, in Escherichia coli. Recombinant PvRII is incorrectly folded and accumulates in inclusion bodies. We have developed methods to refold and purify recombinant PvRII in its functional conformation. Biochemical, biophysical, and functional characterization confirms that recombinant PvRII is pure, homogeneous, and functionally active in that it binds Duffy-positive human erythrocytes with specificity. Refolded PvRII is highly immunogenic and elicits high titer antibodies that can inhibit binding of P. vivax Duffy-binding protein to erythrocytes, providing support for its development as a vaccine candidate for P. vivax malaria. Development of methods to produce functionally active recombinant PvRII is an important step for structural studies as well as vaccine development.
Singh
S
S
Malaria Research Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi 110067, India.
Pandey
K
K
Chattopadhayay
R
R
Yazdani
S S
SS
Lynn
A
A
Bharadwaj
A
A
Ranjan
A
A
Chitnis
C
C
D000595:Amino Acid Sequence; D000818:Animals; D000953:Antigens, Protozoan; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004375:Duffy Blood-Group System / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D004912:Erythrocytes / Q000378:metabolism / Q000469:parasitology; D004926:Escherichia coli; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010966:Plasmodium vivax / Q000378:metabolism*; D011487:Protein Conformation; D017510:Protein Folding; D015800:Protozoan Proteins; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D000953:Antigens, Protozoan; D002352:Carrier Proteins; D004375:Duffy Blood-Group System; C066001:Duffy antigen binding protein, Plasmodium; D010446:Peptide Fragments; D015800:Protozoan Proteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins