Abstract
BackgroundThe Plasmodium vivax Duffy binding protein (PvDBP) has been the most studied ligand binding human reticulocytes to date. This molecule has a cysteine-rich domain in region II (RII) which has been used as control for evaluating the target cell binding activity of several parasite molecules. However, obtaining rPvDBP-RII in a soluble form using the Escherichia coli expression system usually requires laborious and time-consuming steps for recovering the molecule’s structure and function, considering it is extracted from inclusion bodies. The present study describes an easy and fast method for expressing and obtaining several PvDBP fragments which should prove ideal for use in protein–cell interaction assays.ResultsTwo PvDBP encoding regions (rii and riii/v) were cloned in pEXP5-CT vector and expressed in E. coli and extracted from the soluble fraction (rPvDBP-RIIS and rPvDBP-RIII/VS) using a simple freezing/thawing protocol. After the purification, dichroism analysis enabled verifying high rPvDBP-RIIS and rPvDBP-RIII/VS secondary structure α-helix content, which was lowered when molecules were extracted from inclusion bodies (rPvDBP-RIIIB and rPvDBP-RIII/VIB) using a denaturing step. Interestingly, rPvDBP-RIIS, but not rPvDBP-RIIIB, bound to human reticulocytes, while rPvDBP-RIII/VS and rPvDBP-RIII/VIB bound to such cells in a similar way to negative control (cells incubated without recombinant proteins).ConclusionsThis research has shown for the first time how rPvDBP-RII can be expressed and obtained in soluble form using the E. coli system and avoiding the denaturation and refolding steps commonly used. The results highlight the usefulness of the rPvDBP-RIII/VS fragment as a non-binding control for protein-cell target interaction assays. The soluble extraction protocol described is a good alternative to obtain fully functional P. vivax proteins in a fast and easy way, which will surely prove useful to laboratories working in studying this parasite’s biology.
Highlights
The Plasmodium vivax Duffy binding protein (PvDBP) has been the most studied ligand binding human reticulocytes to date
The results showed that Duffy positive human erythrocytes (Duffy+), but not those treated with chymotrypsin, formed rosettes on COS-7 cell surface expressing PvDBP-region II (RII) recombinantly (r); this enabled identifying this domain’s role in binding
Two P. vivax dbp gene regions were selected for amplifying, cloned inside the pEXP5-CT vector and transformed in TOP10 E. coli bacteria; these were pvdbp-rii and pvdbp-riii/v. pvdbp-rii encoding the cysteine-rich domain which can cause erythrocyte rosette formation when expressed on COS-7 cell surface [5]
Summary
The Plasmodium vivax Duffy binding protein (PvDBP) has been the most studied ligand binding human reticulocytes to date. This molecule has a cysteine-rich domain in region II (RII) which has been used as control for evaluating the target cell binding activity of several parasite molecules. The results showed that Duffy positive human erythrocytes (Duffy+), but not those treated with chymotrypsin (which removes DARC), formed rosettes on COS-7 cell surface expressing PvDBP-RII recombinantly (r) (rPvDBP-RII); this enabled identifying this domain’s role in binding. The authors expressed rPvDBP-RII in several fragments (including some Cys residues) on mammal COS cell surface and found that human erythrocytes were capable of forming rosettes only with cells expressing the fragment from Cys 4 to Cys 7 (PvDBP region II). The molecule so purified using a two-step purification protocol by FPLC was able to bind Duffy+ human erythrocytes and induced a natural immune response blocking rPvDBP-RII interaction with cells
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