Production, purification and immunogenicity of a malaria transmission-blocking vaccine candidate: TBV25H expressed in yeast and purified using nickel-NTA agarose.
We have constructed a second generation malaria transmission-blocking vaccine candidate based on Pfs25, the predominate surface protein of Plasmodium falciparum zygotes, to overcome potential production problems with the original construct. Four modifications were made: (1) addition of the last cysteine residue of the fourth epidermal growth factor like-domain of Pfs25; (2) mutagenesis of asparagine-linked glycosylation sites with glutamine rather than alanine; (3) addition of a six histidine tag at the carboxy-terminus for highly efficient purification of recombinant protein on nickel-NTA agarose; and (4) fermentation that combines continuous glucose fed-batch methodology with pH-controlled glucose addition and a terminal ethanol feed. The resulting product, TBV25H (Transmission-Blocking Vaccine based on Pfs25 with a Histidine tag), appears to be a more potent antigen and immunogen than the original construct, and the fermentation and post-fermentation processing methodology easily lend themselves to technology transfer to the ultimate users, newly industrialized countries.
Kaslow
D C
DC
Molecular Vaccine Section, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892.
Shiloach
J
J
D000595:Amino Acid Sequence; D000818:Animals; D000913:Antibodies, Protozoan / Q000097:blood; D015870:Gene Expression; D005822:Genetic Vectors; D017780:Malaria Vaccines / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification; D016778:Malaria, Falciparum / Q000517:prevention & control* / Q000635:transmission; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids; D010963:Plasmodium falciparum / Q000276:immunology*; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D011994:Recombinant Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014614:Vaccines, Synthetic / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification
D000913:Antibodies, Protozoan; D017780:Malaria Vaccines; C071284:Pfs25 protein, Plasmodium falciparum; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D014614:Vaccines, Synthetic; C099774:transmission-Blocking Vaccine based on Pfs25