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Infection and Immunity, April 2008, p. 1702-1708, Vol. 76, No. 4
0019-9567/08/$08.00+0     doi:10.1128/IAI.01539-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Wheat Germ Cell-Free System-Based Production of Malaria Proteins for Discovery of Novel Vaccine Candidates{triangledown} ,{dagger}

Takafumi Tsuboi,1,2* Satoru Takeo,1 Hideyuki Iriko,2,{ddagger} Ling Jin,2 Masateru Tsuchimochi,1 Shusaku Matsuda,1 Eun-Taek Han,1,§ Hitoshi Otsuki,3 Osamu Kaneko,3,¶ Jetsumon Sattabongkot,4 Rachanee Udomsangpetch,5 Tatsuya Sawasaki,1 Motomi Torii,3 and Yaeta Endo1

Cell-Free Science and Technology Research Center,1 Venture Business Laboratory, Ehime University, Matsuyama, Ehime 790-8577, Japan,2 Department of Molecular Parasitology, Ehime University Graduate School of Medicine, Toon, Ehime 791-0295, Japan,3 Department of Entomology, Armed Forces Research Institute of Medical Sciences, Bangkok 10400, Thailand,4 Department of Pathobiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand5

Received 20 November 2007/ Returned for modification 22 December 2007/ Accepted 1 February 2008

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.

* Corresponding author. Mailing address: Cell-Free Science and Technology Research Center, Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan. Phone: 81-89-927-8277. Fax: 81-89-927-9941. E-mail: tsuboi{at}ccr.ehime-u.ac.jp

{triangledown} Published ahead of print on 11 February 2008.

{dagger} Supplemental material for this article may be found at http://iai.asm.org/.

Editor: W. A. Petri, Jr.

{ddagger} Present address: Division of Medical Zoology, Department of Microbiology and Immunology, Faculty of Medicine, Tottori University, Yonago, Tottori 683-8503, Japan.

§ Present address: Department of Parasitology, Kangwon National University College of Medicine, Chunchon 200-701, Korea.

Present address: Department of Protozoology, Institute of Tropical Medicine, Nagasaki University, Nagasaki 852-8523, Japan.

Infection and Immunity, April 2008, p. 1702-1708, Vol. 76, No. 4
0019-9567/08/$08.00+0     doi:10.1128/IAI.01539-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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