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  Community-Wide Experimental Evaluation of the PROSS Stability-Design Method

Peleg, Y., Vincentelli, R., Collins, B. M., Chen, K.-E., Livingstone, E. K., Weeratunga, S., et al. (2021). Community-Wide Experimental Evaluation of the PROSS Stability-Design Method. Journal of Molecular Biology, 433(13): 166964. doi:10.1016/j.jmb.2021.166964.

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 Urheber:
Peleg, Yoav1, Autor
Vincentelli, Renaud1, Autor
Collins, Brett M.1, Autor
Chen, Kai-En1, Autor
Livingstone, Emma K.1, Autor
Weeratunga, Saroja1, Autor
Leneva, Natalya1, Autor
Guo, Qian1, Autor
Remans, Kim1, Autor
Perez, Kathryn1, Autor
Bjerga, Gro E. K.1, Autor
Larsen, Oivind1, Autor
Vanek, Ondrej1, Autor
Skorepa, Ondrej1, Autor
Jacquemin, Sophie1, Autor
Poterszman, Arnaud1, Autor
Kjaer, Svend1, Autor
Christodoulou, Evangelos1, Autor
Albeck, Shira1, Autor
Dym, Orly1, Autor
Ainbinder, Elena1, AutorUnger, Tamar1, AutorSchuetz, Anja1, AutorMatthes, Susann1, AutorBader, Michael1, Autorde Marco, Ario1, AutorStorici, Paola1, AutorSemrau, Marta S.1, AutorStolt-Bergner, Peggy1, AutorAigner, Christian1, AutorSuppmann, Sabine2, Autor           Goldenzweig, Adi1, AutorFleishman, Sarel J.1, Autor mehr..
Affiliations:
1external, ou_persistent22              
2Scientific Service Groups, Max Planck Institute of Biochemistry, Max Planck Society, ou_1565170              

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Schlagwörter: RECOMBINANT PROTEIN EXPRESSION; WEB SERVER; DIRECTED EVOLUTION; R-METHUSCF; TRANSCRIPTION; FILGRASTIM; MUTATIONS; COMPLEX; DOMAIN; TFIIHBiochemistry & Molecular Biology; Protein stability; PROSS; Rosetta; Protein expression; Recombinant proteins;
 Zusammenfassung: Recent years have seen a dramatic improvement in protein-design methodology. Nevertheless, most methods demand expert intervention, limiting their widespread adoption. By contrast, the PROSS algorithm for improving protein stability and heterologous expression levels has been successfully applied to a range of challenging enzymes and binding proteins. Here, we benchmark the application of PROSS as a stand-alone tool for protein scientists with no or limited experience in modeling. Twelve laboratories from the Protein Production and Purification Partnership in Europe (P4EU) challenged the PROSS algorithm with 14 unrelated protein targets without support from the PROSS developers. For each target, up to six designs were evaluated for expression levels and in some cases, for thermal stability and activity. In nine targets, designs exhibited increased heterologous expression levels either in prokaryotic and/or eukaryotic expression systems under experimental conditions that were tailored for each target protein. Furthermore, we observed increased thermal stability in nine of ten tested targets. In two prime examples, the human Stem Cell Factor (hSCF) and human Cadherin-Like Domain (CLD12) from the RET receptor, the wild type proteins were not expressible as soluble proteins in E. coli, yet the PROSS designs exhibited high expression levels in E. coli and HEK293 cells, respectively, and improved thermal stability. We conclude that PROSS may improve stability and expressibility in diverse cases, and that improvement typically requires target-specific expression conditions. This study demonstrates the strengths of community-wide efforts to probe the generality of new methods and recommends areas for future research to advance practically useful algorithms for protein science. (C) 2021 Elsevier Ltd. All rights reserved.

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Sprache(n): eng - English
 Datum: 2021
 Publikationsstatus: Online veröffentlicht
 Seiten: 14
 Ort, Verlag, Ausgabe: -
 Inhaltsverzeichnis: -
 Art der Begutachtung: Expertenbegutachtung
 Identifikatoren: ISI: 000656912400013
DOI: 10.1016/j.jmb.2021.166964
 Art des Abschluß: -

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Titel: Journal of Molecular Biology
  Andere : J Mol Biol
Genre der Quelle: Zeitschrift
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Ort, Verlag, Ausgabe: London : Academic Press
Seiten: - Band / Heft: 433 (13) Artikelnummer: 166964 Start- / Endseite: - Identifikator: ISSN: 0022-2836
CoNE: https://pure.mpg.de/cone/journals/resource/954922646042