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.

Charité University Medicine Charitéplatz 1 10117 Berlin Germany

Department of Biochemistry Faculty of Science Charles University Hlavova 2030 8 12840 Prague Czech Republic

Department of Biomolecular Sciences Weizmann Institute of Science Rehovot 7610001 Israel

Department of Life Sciences Core Facilities Weizmann Institute of Science Rehovot 7610001 Israel

Elettra Sincrotrone Trieste SS 14 km 163 5 in Area Science Park 34149 Basovizza Trieste Italy

European Molecular Biology Laboratory Protein Expression and Purification Core Facility Meyerhofstrasse 1 69117 Heidelberg Germany

German Center for Cardiovascular Research Partner Site Berlin Berlin Germany

Institut de Génétique et de Biologie Moléculaire et Cellulaire U1258 Université de Strasbourg France

Laboratory for Environmental and Life Sciences University of Nova Gorica Slovenia

Max Delbrück Center for Molecular Medicine Robert Rössle Straße 10 13125 Berlin Buch Germany

Max Planck Institute of Biochemistry Biochemistry Core Facility Am Klopferspitz 18 82152 Martinsried Germany

NORCE Norwegian Research Centre Postboks 22 Nygårdstangen 5038 Bergen Norway

Structural Biology Science Technology Platform The Francis Crick Institute 1 Midland Road London NW1 1AT UK

The University of Queensland Institute for Molecular Bioscience St Lucia Queensland 4072 Australia

Unité Mixte de Recherche Marseille France

University of Lübeck Institute for Biology Ratzeburger Allee 160 23562 Lübeck Germany

Vienna Biocenter Core Facilities GmbH Dr Bohr gasse 3 1030 Vienna Austria

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