Abstract
Appropriate resources and expression technology necessary for human proteomics on a whole-proteome scale are being developed. We prepared a foundation for simple and efficient production of human proteins using the versatile Gateway vector system. We generated 33,275 human Gateway entry clones for protein synthesis, developed mRNA expression protocols for them and improved the wheat germ cell-free protein synthesis system. We applied this protein expression system to the in vitro expression of 13,364 human proteins and assessed their biological activity in two functional categories. Of the 75 tested phosphatases, 58 (77%) showed biological activity. Several cytokines containing disulfide bonds were produced in an active form in a nonreducing wheat germ cell-free expression system. We also manufactured protein microarrays by direct printing of unpurified in vitro–synthesized proteins and demonstrated their utility. Our 'human protein factory' infrastructure includes the resources and expression technology for in vitro proteome research.
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Acknowledgements
This work was supported by the grant 'Functional Analysis of Human Proteins and Its Research Application' from the New Energy and Industrial Technology Development Organization (NEDO), Japan. We thank the Research Association for Biotechnology and Helix Research Institute for providing FLJ cDNA clones, S. Sugano (Graduate School of Frontier Sciences, The University of Tokyo) for providing the oligo-capped cDNA libraries, helpful suggestions and encouragement during the work, A. Miyawaki (Brain Science Institute, RIKEN) for providing the plasmid encoding the Venus protein, and S. Ebisu (ProteinExpress, Co., Ltd.), H. Takakura (Takara) and M. Hirano (Toray Research Center, Inc.) for providing E. coli cells for protein expression systems.
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Contributions
N.G., Yo.K, Ya.K., S.T., Ka.M., A.W., T.I., S.W. and N.N. designed and supervised the project. Ay.M., Yo.K., J.-i.I., N.G. and N.N. wrote the manuscript. T.I., A.W., J.-i.Y., Ko.K., T.N., T.A. and R.K. determined ORF regions and selected templates. T.A., N.K., R.K., K.I, At.M., T.O., Ki.K., Yuk.S.,Ts.S., S.S., Ya.K., K.Y., N.S. and S.T. produced Gateway entry clones. Y.I., K.-i.K., Y.T. and Ki.M. sequenced Gateway entry clones. K.Y., R.S., Y.M., C.K. and N.S. re-examined identity and stability of Gateway entry clones at the final step. B.K, A.S., T.K., Mar.M., H.T. and N.K. produced Gateway destination vectors. R.M., Ay.M., A.F., Yut.S., To.S., Mas.M., To.T., H.K. and H.T. performed preliminary analysis for determining best conditions for protein production and other experimental protocols. A.F., Yut.S., H.Y., B.K., A.S., T.K., S.N. and M.K. expressed proteins and performed SDS-PAGE. A.F., To.S., To.T., Mas.M. and Yo.K. analyzed the SDS-PAGE data. A.F., Ay.M. and Yo.K. analyzed phosphatase activity. Ay.M., Ta.T., R.H., Y.Y., E.I., A.N. and J.-i.I. analyzed cytokine activity by DNA microarrays. A.F., H.Y., C.K., Yut.S, Yo.K. and J.-i.I. produced protein microarrays. A.F., Yo.K. and J.-i.I. analyzed data of protein microarrays. F.I. and Y.E. made comments and encouraged the idea.
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Ko.K. and T.N. are employees of Hitachi. Y.I., K.-i.K. and K.Y. are employees of Hitachi Science Systems. B.K. and T.K. are employees of Toyobo. M.K. is an employee of Mitsubishi Chemical Group. S.N. is an employee of Wakenyaku.
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Supplementary Text and Figures
Supplementary Figures 1–11, Supplementary Tables 1, 3–7 and 9, Supplementary Methods, Supplementary Note (PDF 3498 kb)
Supplementary Table 2
Features of all Gateway standard entry and its source cDNA clones. (TXT 2162 kb)
Supplementary Table 8
List of 13,277 Gateway entry clones whose proteins were mounted on protein microarrays and fluorescence intensity of each spot. (TXT 865 kb)
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Goshima, N., Kawamura, Y., Fukumoto, A. et al. Human protein factory for converting the transcriptome into an in vitro–expressed proteome. Nat Methods 5, 1011–1017 (2008). https://doi.org/10.1038/nmeth.1273
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DOI: https://doi.org/10.1038/nmeth.1273
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