Wheat germ cell-free system-based production of malaria proteins for discovery of novel vaccine candidates.
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.
Tsuboi
Takafumi
T
Cell-Free Science and Technology Research Center, Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan. tsuboi@ccr.ehime-u.ac.jp
Takeo
Satoru
S
Iriko
Hideyuki
H
Jin
Ling
L
Tsuchimochi
Masateru
M
Matsuda
Shusaku
S
Han
Eun-Taek
ET
Otsuki
Hitoshi
H
Kaneko
Osamu
O
Sattabongkot
Jetsumon
J
Udomsangpetch
Rachanee
R
Sawasaki
Tatsuya
T
Torii
Motomi
M
Endo
Yaeta
Y
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
D000953:Antigens, Protozoan; D017780:Malaria Vaccines; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D014614:Vaccines, Synthetic