title: Bacterially expressed truncated F2 domain of Plasmodium falciparum EBA-140 antigen can bind to human erythrocytes.
abstract: The recently identified erythrocyte binding antigen-140 (EBA-140) is a member of the Plasmodium falciparum DBL family of erythrocyte binding proteins, which are considered as prospective candidates for malaria vaccine development. The EBA-140 ligand is a paralogue of the well-characterized P. falciparum EBA-175 antigen. They share homology of domain structure, including Region II, which consists of two homologous F1 and F2 domains and is responsible for ligand-erythrocyte interaction during invasion. It was shown that the F2 domain of EBA-175 antigen seems to be more important for erythrocyte binding. In order to study activity and immunogenicity of EBA-140 antigen F2 domain, it is necessary to obtain recombinant protein of high purity and in a sufficient amount, which used to pose a challenge due to the high content of disulphide bridges. Here, we present a new method for expression and purification of Plasmodium falciparum EBA-140 antigen F2 domain in E. coli Rosetta-gami strain in fusion with the maltose binding protein (MBP). The truncated F2 domain formed by spontaneous proteolytic degradation of the fusion protein was purified by affinity chromatography on Ni-NTA resin followed by size exclusion chromatography. Molecular mass of this protein was confirmed by mass spectrometry. Its N-terminal amino acid sequencing revealed a proteolytic cleavage site within the F2 domain. The proper folding of the recombinant, truncated F2 domain of EBA-140 antigen was confirmed by circular dichroism analysis. The truncated F2 domain can specifically bind to human erythrocytes but its binding is not as efficient as that of full Region II. This confirms that both the F1 and F2 domains of EBA-140 antigen are required for effective erythrocyte binding.
authors.affiliation: Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland. jaskiew@iitd.pan.wroc.pl
authors.forename: Joanna
authors.initials: J
authors.lastname: Rydzak
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Kryńska
authors.forename: Anna
authors.initials: A
authors.lastname: Suchanowska
authors.forename: Radosław
authors.initials: R
authors.lastname: Kaczmarek
authors.forename: Jolanta
authors.initials: J
authors.lastname: Łukasiewicz
authors.forename: Marcin
authors.initials: M
authors.lastname: Czerwiński
authors.forename: Ewa
authors.initials: E
authors.lastname: Jaśkiewicz
mesh_terms: D000953:Antigens, Protozoan / Q000235:genetics / Q000276:immunology; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics; D004912:Erythrocytes / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli; D006801:Humans; D016778:Malaria, Falciparum / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins; D010963:Plasmodium falciparum / Q000737:chemistry / Q000378:metabolism / Q000472:pathogenicity; D011446:Prospective Studies; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D000953:Antigens, Protozoan; D002352:Carrier Proteins; C438380:EBA140 protein, Plasmodium falciparum; D008565:Membrane Proteins; D015800:Protozoan Proteins; D011994:Recombinant Proteins