title: Insect cells are superior to Escherichia coli in producing malaria proteins inducing IgG targeting PfEMP1 on infected erythrocytes.
abstract: BACKGROUND
  The PFD1235w Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) antigen is associated with severe malaria in children and can be expressed on the surface of infected erythrocytes (IE) adhering to ICAM1. However, the exact three-dimensional structure of this PfEMP1 and its surface-exposed epitopes are unknown. An insect cell and Escherichia coli based system was used to express single and double domains encoded by the pfd1235w var gene. The resulting recombinant proteins have been evaluated for yield and purity and their ability to induce rat antibodies, which react with the native PFD1235w PfEMP1 antigen expressed on 3D7PFD1235w-IE. Their recognition by human anti-malaria antibodies from previously infected Tanzanian donors was also analysed.
  
  
  METHODS
  The recombinant proteins were run on SDS-PAGE and Western blots for quantification and size estimation. Insect cell and E. coli-produced recombinant proteins were coupled to a bead-based Luminex assay to measure the plasma antibody reactivity of 180 samples collected from Tanzanian individuals. The recombinant proteins used for immunization of rats and antisera were also tested by flow cytometry for their ability to surface label 3D7PFD1235w-IE.
  
  
  RESULTS
  All seven pAcGP67A constructs were successfully expressed as recombinant protein in baculovirus-infected insect cells and subsequently produced to a purity of 60-97% and a yield of 2-15 mg/L. By comparison, only three of seven pET101/D-TOPO constructs expressed in the E. coli system could be produced at all with purity and yield ranging from 3-95% and 6-11 mg/L. All seven insect cell, but only two of the E. coli produced proteins induced antibodies reactive with native PFD1235w expressed on 3D7PFD1235w-IE. The recombinant proteins were recognized in an age- and transmission intensity-dependent manner by antibodies from 180 Tanzanian individuals in a bead-based Luminex assay.
  
  
  CONCLUSIONS
  The baculovirus based insect cell system was distinctly superior to the E. coli expression system in producing a larger number of different recombinant PFD1235w protein domains and these were significantly easier to purify at a useful yield. However, proteins produced in both systems were able to induce antibodies in rats, which can recognize the native PFD1235w on the surface of IE.
authors.affiliation: Department of International Health, University of Copenhagen, Copenhagen K, Denmark.
authors.forename: Michala E
authors.initials: ME
authors.lastname: Victor
authors.forename: Anja
authors.initials: A
authors.lastname: Bengtsson
authors.forename: Gorm
authors.initials: G
authors.lastname: Andersen
authors.forename: Dominique
authors.initials: D
authors.lastname: Bengtsson
authors.forename: John P
authors.initials: JP
authors.lastname: Lusingu
authors.forename: Lasse S
authors.initials: LS
authors.lastname: Vestergaard
authors.forename: David E
authors.initials: DE
authors.lastname: Arnot
authors.forename: Thor G
authors.initials: TG
authors.lastname: Theander
authors.forename: Louise
authors.initials: L
authors.lastname: Joergensen
authors.forename: Anja T R
authors.initials: AT
authors.lastname: Jensen
mesh_terms: D000293:Adolescent; D000818:Animals; D000913:Antibodies, Protozoan / Q000097:blood*; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D002648:Child; D002675:Child, Preschool; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood*; D007313:Insecta; D017780:Malaria Vaccines / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D016778:Malaria, Falciparum / Q000276:immunology* / Q000517:prevention & control*; D015800:Protozoan Proteins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D051381:Rats; D013636:Tanzania; D014614:Vaccines, Synthetic / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D055815:Young Adult
chemical_list: D000913:Antibodies, Protozoan; D007074:Immunoglobulin G; D017780:Malaria Vaccines; D015800:Protozoan Proteins; D014614:Vaccines, Synthetic; C085731:erythrocyte membrane protein 1, Plasmodium falciparum