Summary
Expression-ready cDNA clones, where the open reading frame (ORF) of the gene of interest is placed under the control of an appropriate promoter, are critical for functional characterization of the gene products. To create a resource of human gene products, we attempted to systematically convert original cDNA clones to expression-ready forms for recombinant proteins. For this purpose, we adopted a rare-cutting restriction enzyme-based system, the Flexi® cloning system, to construct ORF clones. Taking advantage of the fully sequenced cDNA clones we accumulated to date, a number of sets of Flexi® ORF clones in a 96-well format have been prepared. In this section, two methods for the preparation of Flexi® ORF clones in a 96-well format are described. A protocol for transferring ORFs between Flexi® vectors in a 96-well format is also described. We believe that the resultant clone set could be successfully used as a versatile reagent for functional characterization of human proteins.
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Acknowledgments
I would like to thank T. Watanabe, M. Tamura, K. Yamada, E. Suzuki, C. Mori, and H. Kinoshita for their technical assistance. The Kazusa ORFeome Project of which this report is a part is organized by Dr. O. Ohara and Dr. T. Nagase (Kazusa DNA Research Institute). This work is supported in part by a grant from the Promega Corporation and by a special grant from the Chiba Prefectural Government for acceleration of the practical application of biotechnology.
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Yamakawa, H. (2009). High-Throughput Construction of ORF Clones for Production of the Recombinant Proteins. In: Koga, H. (eds) Reverse Chemical Genetics. Methods in Molecular Biology™, vol 577. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-232-2_3
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DOI: https://doi.org/10.1007/978-1-60761-232-2_3
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