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

Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved..

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.

Medienart:

E-Artikel

Erscheinungsjahr:

2020

Erschienen:

2020

Enthalten in:

Zur Gesamtaufnahme - volume:58

Enthalten in:

New biotechnology - 58(2020) vom: 25. Sept., Seite 45-54

Sprache:

Englisch

Beteiligte Personen:

Tegel, Hanna [VerfasserIn]
Dannemeyer, Melanie [VerfasserIn]
Kanje, Sara [VerfasserIn]
Sivertsson, Åsa [VerfasserIn]
Berling, Anna [VerfasserIn]
Svensson, Anne-Sophie [VerfasserIn]
Hober, Andreas [VerfasserIn]
Enstedt, Henric [VerfasserIn]
Volk, Anna-Luisa [VerfasserIn]
Lundqvist, Magnus [VerfasserIn]
Moradi, Mona [VerfasserIn]
Afshari, Delaram [VerfasserIn]
Ekblad, Siri [VerfasserIn]
Xu, LanLan [VerfasserIn]
Westin, Malin [VerfasserIn]
Bidad, Faranak [VerfasserIn]
Schiavone, Lovisa Holmberg [VerfasserIn]
Davies, Rick [VerfasserIn]
Mayr, Lorenz M [VerfasserIn]
Knight, Sinead [VerfasserIn]
Göpel, Sven O [VerfasserIn]
Voldborg, Björn G [VerfasserIn]
Edfors, Fredrik [VerfasserIn]
Forsström, Björn [VerfasserIn]
von Feilitzen, Kalle [VerfasserIn]
Zwahlen, Martin [VerfasserIn]
Rockberg, Johan [VerfasserIn]
Takanen, Jenny Ottosson [VerfasserIn]
Uhlén, Mathias [VerfasserIn]
Hober, Sophia [VerfasserIn]

Links:

Volltext

Themen:

9007-49-2
DNA
High-throughput
Journal Article
Protein production
Protein purification
Recombinant Proteins
Secreted proteins

Anmerkungen:

Date Completed 14.12.2020

Date Revised 14.12.2020

published: Print-Electronic

Citation Status MEDLINE

doi:

10.1016/j.nbt.2020.05.002

funding:

Förderinstitution / Projekttitel:

PPN (Katalog-ID):

NLM310821789