Heterologous expression of Plasmodium vivax apical membrane antigen 1 (PvAMA1) for binding peptide selection

Abstract

BackgroundPlasmodium is an obligate intracellular parasite. Apical membrane antigen 1 (AMA1) is the most prominent and well characterized malarial surface antigen that is essential for parasite-host cell invasion, i.e., for sporozoite to invade and replicate within hepatocytes in the liver stage and merozoite to penetrate and replicate within erythrocytes in the blood stage. AMA1 has long served as a potent antimalarial drug target and is a pivotal vaccine candidate. A good understanding of the structure and molecular function of this Plasmodium protein, particularly its involvement in host-cell adhesion and invasion, is of great interest and hence it offers an attractive target for the development of novel therapeutics. The present study aims to heterologous express recombinant Plasmodium AMA1 ectodomain of P. vivax (rPvAMA1) for the selection of binding peptides.MethodsThe rPvAMA1 protein was heterologous expressed using a tag-free Profinity eXactTM system and codon optimized BL21-Codon Plus (DE3)-RIL Escherichia coli strain and further refolded by dialysis for renaturation. Binding peptides toward refolded rPvAMA1 were panned using a Ph.D.-12 random phage display library.ResultsThe rPvAMA1 was successfully expressed and refolded with three phage-displayed dodecapeptides designated as PdV1 (DLTFTVNPLSKA), PdV2 (WHWSWWNPNQLT), and PdV3 (TSVSYINNRHNL) with affinity towards rPvAMA1 identified. All of them exhibited positive binding signal to rPvAMA1 in both direct phage assays, i.e., phage ELISA binding assay and Western blot binding assay.DiscussionPhage display technology enables the mapping of protein-protein interactions based on a simple principle that a library of phage particles displaying peptides is used and the phage clones that bind to the target protein are selected and identified. The binding sites of each selected peptides toward PvAMA1 (Protein Data Bank, PDB ID: 1W8K) were in silico predicted using CABS-dock web server. In this case, the binding peptides provide a valuable starting point for the development of peptidomimetic as antimalarial antagonists directed at PvAMA1.

Development of a Phage Display Panning Strategy Utilizing Crude Antigens: Isolation of MERS-CoV Nucleoprotein human antibodies

“…The main assurance is that these proteins are mainly prepared with good purity and yield. This is evident with the reports of antibody development for dengue virus 10 , herpes simplex virus 11 , cytomegalovirus 11 , Zika virus 12 , Ebola virus 7,13 , Plasmodium species 14 , Mycobacterium tuberculosis 15 , and nematodes responsible for filariasis 16 .…”

Identification of Plasmodium knowlesi Merozoite Surface Protein‐119 (PkMSP‐119) novel binding peptides from a phage display library

“…Computational docking methods have proved useful in predicting protein-peptide interactions and help in the identification of peptide binding sites [49,50]. For example, rPvAMA-1 potential binding peptides selected from phage display libraries have been mapped to the location on the protein ectoplasmic region and the binding sites were successfully predicted by using CABS-dock [39]. In our study, the binding sites of phage display-screened peptides towards PkMSP-1 19 were in silico predicted by using CABS-dock web server.…”

“…After running the protocol, CABS-dock server-generated top 10 best-ranked simulation models, and the quality of each docking model was recorded in the web server. The quality of the docking models was assessed using ligand root-mean-square deviation (RMSD) as follows: RMSD < 3 A indicates high-quality prediction; 3 A ≤ RMSD ≤ 5.5 A medium-quality prediction; and RMSD > 5.5 A low-quality prediction [39]. In the present study, only one of the top-ranked models was analysed and reported for each of the binding peptide docking protocol.…”

Immunoinformatic approach to assess SARS-CoV-2 protein S epitopes recognised by the most frequent MHC-I alleles in the Brazilian population

“…After the docking, we excluded the complexes that showed root mean squared deviation (RMSD) values above 3 Å to follow the molecular dynamics (MD). 23…”