Wheat Germ Cell-Free System-Based Production of Malaria Proteins for Discovery of Novel Vaccine Candidates
| Autor: | Shusaku Matsuda, Eun-Taek Han, Yaeta Endo, Hitoshi Otsuki, Hideyuki Iriko, Rachanee Udomsangpetch, Ling Jin, Masateru Tsuchimochi, Osamu Kaneko, Jetsumon Sattabongkot, Satoru Takeo, Takafumi Tsuboi, Motomi Torii, Tatsuya Sawasaki |
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| Rok vydání: | 2008 |
| Předmět: |
Plasmodium falciparum
Immunology Protozoan Proteins Antigens Protozoan Microbiology law.invention Mice law Malaria Vaccines parasitic diseases Protein biosynthesis Gametocyte Animals Humans Gene Triticum Vaccines Synthetic Cell-Free System biology Malaria vaccine Immunogenicity biology.organism_classification Virology Recombinant Proteins Culicidae Infectious Diseases Protein Biosynthesis Codon usage bias Recombinant DNA Parasitology Fungal and Parasitic Infections |
| Zdroj: | Infection and Immunity. 76:1702-1708 |
| ISSN: | 1098-5522 0019-9567 |
| DOI: | 10.1128/iai.01539-07 |
| Popis: | 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 μg/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. |
| Databáze: | OpenAIRE |
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