title: Cell-Free Expression and In Situ Immobilization of Parasite Proteins from Clonorchis sinensis for Rapid Identification of Antigenic Candidates.
abstract: Progress towards genetic sequencing of human parasites has provided the groundwork for a post-genomic approach to develop novel antigens for the diagnosis and treatment of parasite infections. To fully utilize the genomic data, however, high-throughput methodologies are required for functional analysis of the proteins encoded in the genomic sequences. In this study, we investigated cell-free expression and in situ immobilization of parasite proteins as a novel platform for the discovery of antigenic proteins. PCR-amplified parasite DNA was immobilized on microbeads that were also functionalized to capture synthesized proteins. When the microbeads were incubated in a reaction mixture for cell-free synthesis, proteins expressed from the microbead-immobilized DNA were instantly immobilized on the same microbeads, providing a physical linkage between the genetic information and encoded proteins. This approach of in situ expression and isolation enables streamlined recovery and analysis of cell-free synthesized proteins and also allows facile identification of the genes coding antigenic proteins through direct PCR of the microbead-bound DNA.
authors.affiliation: Department of Fine Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon 305-764, Korea.
authors.forename: Christy
authors.initials: C
authors.lastname: Catherine
authors.affiliation: Department of Fine Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon 305-764, Korea.
authors.forename: Seung-Won
authors.initials: SW
authors.lastname: Lee
authors.affiliation: Division of Malaria and Parasitic Diseases, National Institute of Health, Osong 361-951, Korea.
authors.forename: Jung Won
authors.initials: JW
authors.lastname: Ju
authors.affiliation: Department of Fine Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon 305-764, Korea.
authors.forename: Ho-Cheol
authors.initials: HC
authors.lastname: Kim
authors.affiliation: Division of Malaria and Parasitic Diseases, National Institute of Health, Osong 361-951, Korea.
authors.forename: Hyun-Il
authors.initials: HI
authors.lastname: Shin
authors.affiliation: Division of Malaria and Parasitic Diseases, National Institute of Health, Osong 361-951, Korea.
authors.forename: Yu Jung
authors.initials: YJ
authors.lastname: Kim
authors.affiliation: Department of Fine Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon 305-764, Korea.
authors.forename: Dong-Myung
authors.initials: DM
authors.lastname: Kim
mesh_terms: D000818:Animals; D000947:Antigens, Helminth / Q000276:immunology*; D002474:Cell-Free System; D003004:Clonorchis sinensis / Q000276:immunology* / Q000378:metabolism*; D004247:DNA / Q000378:metabolism
chemical_list: D000947:Antigens, Helminth; D004247:DNA