Cell-Free Expression and In Situ Immobilization of Parasite Proteins from Clonorchis sinensis for Rapid Identification of Antigenic Candidates |
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Authors: | Christy Catherine Seung-Won Lee Jung Won Ju Ho-Cheol Kim Hyun-Il Shin Yu Jung Kim Dong-Myung Kim |
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Affiliation: | 1. Department of Fine Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon 305–764, Korea.; 2. Division of Malaria and Parasitic Diseases, National Institute of Health, Osong 361–951, Korea.; University of Saskatchewan, CANADA, |
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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. |
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