High throughput generation of a resource of the human secretome in mammalian cells,New Biotechnology

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High throughput generation of a resource of the human secretome in mammalian cells
New Biotechnology ( IF 5.4 ) Pub Date : 2020-09-01 , DOI: 10.1016/j.nbt.2020.05.002
Hanna Tegel ₁ , Melanie Dannemeyer ₁ , Sara Kanje ₁ , Åsa Sivertsson ₂ , Anna Berling ₁ , Anne-Sophie Svensson ₁ , Andreas Hober ₂ , Henric Enstedt ₁ , Anna-Luisa Volk ₁ , Magnus Lundqvist ₁ , Mona Moradi ₁ , Delaram Afshari ₁ , Siri Ekblad ₁ , LanLan Xu ₁ , Malin Westin ₁ , Faranak Bidad ₁ , Lovisa Holmberg Schiavone ₃ , Rick Davies ₄ , Lorenz M Mayr ₅ , Sinead Knight ₄ , Sven O Göpel ₆ , Björn G Voldborg ₇ , Fredrik Edfors ₂ , Björn Forsström ₂ , Kalle von Feilitzen ₂ , Martin Zwahlen ₂ , Johan Rockberg ₁ , Jenny Ottosson Takanen ₁ , Mathias Uhlén ₈ , Sophia Hober ₁

Affiliation

Department of Protein Science, School of Chemistry, Biotechnology and Health, KTH - Royal Institute of Technology, Stockholm, Sweden.
Department of Protein Science, School of Chemistry, Biotechnology and Health, KTH - Royal Institute of Technology, Stockholm, Sweden; Science for Life Laboratory, KTH - Royal Institute of Technology, Stockholm, Sweden.
Discovery Biology, Discovery Sciences, R&D, AstraZeneca, Gothenburg, Sweden.
Discovery Biology, Discovery Sciences, R&D, AstraZeneca, Cambridge, UK.
Syncona Investment Ltd, London, UK; ETH Zurich, B-BIOL, Molecular Health Sciences, Zurich, Switzerland.
Bioscience Metabolism, Research and Early Development, Cardiovascular, Renal and Metabolism, BioPharmaceutical R&D, AstraZeneca, Gothenburg, Sweden.
Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Denmark.
Department of Protein Science, School of Chemistry, Biotechnology and Health, KTH - Royal Institute of Technology, Stockholm, Sweden; Science for Life Laboratory, KTH - Royal Institute of Technology, Stockholm, Sweden; Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Denmark.

The proteins secreted by human tissues and blood cells, the secretome, are important both for the basic understanding of human biology and for identification of potential targets for future diagnosis and therapy. Here, a high-throughput mammalian cell factory is presented that was established to create a resource of recombinant full-length proteins covering the majority of those annotated as 'secreted' in humans. The full-length DNA sequences of each of the predicted secreted proteins were generated by gene synthesis, the constructs were transfected into Chinese hamster ovary (CHO) cells and the recombinant proteins were produced, purified and analyzed. Almost 1,300 proteins were successfully generated and proteins predicted to be secreted into the blood were produced with a success rate of 65%, while the success rates for the other categories of secreted proteins were somewhat lower giving an overall one-pass success rate of ca. 58%. The proteins were used to generate targeted proteomics assays and several of the proteins were shown to be active in a phenotypic assay involving pancreatic β-cell dedifferentiation. Many of the proteins that failed during production in CHO cells could be rescued in human embryonic kidney (HEK 293) cells suggesting that a cell factory of human origin can be an attractive alternative for production in mammalian cells. In conclusion, a high-throughput protein production and purification system has been successfully established to create a unique resource of the human secretome.

更新日期：2020-09-01

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