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Community-wide experimental evaluation of the PROSS stability-design method

Item Type:Article
Title:Community-wide experimental evaluation of the PROSS stability-design method
Creators Name:Peleg, Y. and Vincentelli, R. and Collins, B.M. and Chen, K.E. and Livingstone, E.K. and Weeratunga, S. and Leneva, N. and Guo, Q. and Remans, K. and Perez, K. and Bjerga, G.E.K. and Larsen, Ø. and Vaněk, O. and Skořepa, O. and Jacquemin, S. and Poterszman, A. and Kjaer, S. and Christodoulou, E. and Albeck, S. and Dym, O. and Ainbinder, E. and Unger, T. and Schuetz, A. and Matthes, S. and Bader, M. and de Marco, A. and Storici, P. and Semrau, M.S. and Stolt-Bergner, P. and Aigner, C. and Suppmann, S. and Goldenzweig, A. and Fleishman, S.J.
Abstract: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. Up to six designs were evaluated in each case for expression, stability, and, in some cases, 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.
Keywords:Protein Stability, PROSS, Rosetta, Protein Expression, Recombinant Proteins, Animals, Zebrafish
Source:Journal of Molecular Biology
Page Range:166964
Date:25 June 2021
Official Publication:https://doi.org/10.1016/j.jmb.2021.166964
PubMed:View item in PubMed

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