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Easy and fast method for expression and native extraction of Plasmodium vivax Duffy binding protein fragments.

ABSTRACT: BACKGROUND: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. 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. RESULTS:Two 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 ?-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). CONCLUSIONS:This 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.

SUBMITTER: Moreno-Perez DA

PROVIDER: S-EPMC5806264 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Easy and fast method for expression and native extraction of Plasmodium vivax Duffy binding protein fragments.

Moreno-Pérez Darwin Andrés DA Baquero Luis Alfredo LA Bermúdez Maritza M Gómez-Muñoz Laura Alejandra LA Varela Yahson Y Patarroyo Manuel Alfonso MA

Malaria journal 20180208 1

<h4>Background</h4>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. 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 f ...[more]

PMID: 29422046

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Project description:BackgroundThe Duffy-binding protein II of Plasmodium vivax (PvDBPII) has been considered as an attractive target for vaccine-mediated immunity despite a possible highly polymorphic nature. Among seven PvDBP domains, domain II has been shown to exhibit a high rate of nonsynonymous polymorphism, which has been suggested to be a potential immune (antibody binding) evasion mechanism. This study aimed to determine the extent of genetic polymorphisms and positive natural selection at domain II of the PvDBP gene among a sampling of Thai P. vivax isolates.MethodsThe PvDBPII gene was PCR amplified and the patterns of polymorphisms were characterized from 30 Thai P. vivax isolates using DNA cloning and sequencing. Phylogenetic analysis of the sequences and positive selection were done using DnaSP ver 4.0 and MEGA ver 4.0 packages.ResultsThis study demonstrated a high rate of nonsynonymous polymorphism. Using Sal I as the reference strain, a total of 30 point-mutations were observed in the PvDBPII gene among the set of Thai P. vivax isolates, of which 25 nonsynonymous and five synonymous were found. The highest frequency of polymorphism was found in five variant amino acids (residues D384G, R390H, L424I, W437R, I503K) with the variant L424I having the highest frequency. The difference between the rates of nonsynonymous and synonymous mutations estimated by the Nei and Gojobori's method suggested that PvDBPII antigen appears to be under selective pressure. Phylogenetic analysis of PvDBPII Thai P. vivax isolates to others found internationally demonstrated six distinct allele groups. Allele groups 4 and 6 were unique to Thailand.ConclusionPolymorphisms within PvDBPII indicated that Thai vivax malaria parasites are genetically diverse. Phylogenetic analysis of DNA sequences using the Neighbour-Joining method demonstrated that Thai isolates shared distinct alleles with P. vivax isolates from different geographical areas. The study reported here will be valuable for the development of PvDBPII-based malaria vaccine.

Dataset Information

Easy and fast method for expression and native extraction of Plasmodium vivax Duffy binding protein fragments.

Publications

Easy and fast method for expression and native extraction of Plasmodium vivax Duffy binding protein fragments.

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