title: Wheat germ cell-free system-based production of malaria proteins for discovery of novel vaccine candidates.
abstract: One of the major bottlenecks in malaria research has been the difficulty in recombinant protein expression. Here, we report the application of the wheat germ cell-free system for the successful production of malaria proteins. For proof of principle, the Pfs25, PfCSP, and PfAMA1 proteins were chosen. These genes contain very high A/T sequences and are also difficult to express as recombinant proteins. In our wheat germ cell-free system, native and codon-optimized versions of the Pfs25 genes produced equal amounts of proteins. PfCSP and PfAMA1 genes without any codon optimization were also expressed. The products were soluble, with yields between 50 and 200 mug/ml of the translation mixture, indicating that the cell-free system can be used to produce malaria proteins without any prior optimization of their biased codon usage. Biochemical and immunocytochemical analyses of antibodies raised in mice against each protein revealed that every antibody retained its high specificity to the parasite protein in question. The development of parasites in mosquitoes fed patient blood carrying Plasmodium falciparum gametocytes and supplemented with our mouse anti-Pfs25 sera was strongly inhibited, indicating that both Pfs25-3D7/WG and Pfs25-TBV/WG retained their immunogenicity. Lastly, we carried out a parallel expression assay of proteins of blood-stage P. falciparum. The PCR products of 124 P. falciparum genes chosen from the available database were used directly in a small-scale format of transcription and translation reactions. Autoradiogram testing revealed the production of 93 proteins. The application of this new cell-free system-based protocol for the discovery of malaria vaccine candidates will be discussed.
authors.affiliation: Cell-Free Science and Technology Research Center, Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan. tsuboi@ccr.ehime-u.ac.jp
authors.forename: Takafumi
authors.initials: T
authors.lastname: Tsuboi
authors.forename: Satoru
authors.initials: S
authors.lastname: Takeo
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Iriko
authors.forename: Ling
authors.initials: L
authors.lastname: Jin
authors.forename: Masateru
authors.initials: M
authors.lastname: Tsuchimochi
authors.forename: Shusaku
authors.initials: S
authors.lastname: Matsuda
authors.forename: Eun-Taek
authors.initials: ET
authors.lastname: Han
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Otsuki
authors.forename: Osamu
authors.initials: O
authors.lastname: Kaneko
authors.forename: Jetsumon
authors.initials: J
authors.lastname: Sattabongkot
authors.forename: Rachanee
authors.initials: R
authors.lastname: Udomsangpetch
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Sawasaki
authors.forename: Motomi
authors.initials: M
authors.lastname: Torii
authors.forename: Yaeta
authors.initials: Y
authors.lastname: Endo
mesh_terms: D000818:Animals; D000953:Antigens, Protozoan; D002474:Cell-Free System / Q000737:chemistry*; D009033:Culicidae; D006801:Humans; D017780:Malaria Vaccines; D051379:Mice; D010963:Plasmodium falciparum / Q000378:metabolism*; D014176:Protein Biosynthesis; D015800:Protozoan Proteins / Q000737:chemistry*; D011994:Recombinant Proteins; D014908:Triticum; D014614:Vaccines, Synthetic
chemical_list: D000953:Antigens, Protozoan; D017780:Malaria Vaccines; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D014614:Vaccines, Synthetic