aalto1 untyped-item.component.html
Exploring condensate properties of engineered proteins in cellular and in vitro systems for materials design
Loading...
URL
Journal Title
Journal ISSN
Volume Title
School of Chemical Engineering |
Doctoral thesis (article-based)
| Defence date: 2025-10-10
Electronic archive copy is available via Aalto Thesis Database.
Unless otherwise stated, all rights belong to the author. You may download, display and print this publication for Your own personal use. Commercial use is prohibited.
Authors
Date
Major/Subject
Mcode
Degree programme
Language
en
Pages
80 + app. 158
Series
Aalto University publication series Doctoral Theses, 199/2025
Abstract
Biological materials have long been a source of inspiration for developing high-performance synthetic materials. There is a particular interest in developing biomimetic materials using proteins, the building blocks of many biological materials. Increasing evidence shows that liquid-liquid phase separation (LLPS) is an intermediate assembly step for materials like spider silk and mussel adhesive. LLPS enables the formation of liquid condensates, i.e., protein-dense phases, allowing pre-organization of proteins before transitioning into solid materials. This suggests LLPS proteins derived from biomaterials can be harnessed to make biosynthetic materials. However, many of these proteins need to be redesigned to enable their soluble production in heterologous hosts (e.g., E. coli) and to undergo LLPS in non-biological systems. The molecular mechanisms behind engineering condensates and tuning their properties for material design are not yet fully understood. Given that LLPS also drives the formation of intracellular biomolecular condensates, which are wellcharacterized, we explored whether cells could function as living test tubes for identifying and characterizing engineered protein condensates. Reliable intracellular characterizations could facilitate protein design for functional materials. In this thesis, we worked toward this goal and highlighted critical aspects that need to be considered when using intracellular characterizations for screening condensating proteins for materials development.
In publication 1, the complexity of intracellular behaviors of protein was understood with a model endogenous protein, Sup35, in Saccharomyces cerevisiae. Sup35 can form two distinct structures: reversible condensates in the acidic cytoplasm, and irreversible aggregates upon Sup35 overexpression. Sup35 aggregates were associated with cell growth inhibition. The complete aggregation of Sup35 caused by very high concentrations of Sup35 can diminish Sup35 condensation and impair cellular recovery from stress.
In publications 2 and 3, the assembly of a spidroin NT2RepCT, were compared in different systems, i.e., Saccharomyces cerevisiae, E. coli, and in vitro. In E. coli, the protein formed liquid condensates that can grow larger with increasing concentrations, consistently with its behaviors in vitro. The protein formed multiple small condensates that did not grow significantly with increasing concentrations in yeast cells, but the coalescence of condensates was observed immediately after decreasing cytosolic pH of yeast cells, recapitulating a key feature of NT2RepCT which undergoes low pH-induced liquid-to-solid transition into fibrils in vitro. These suggested different systems might affect condensate properties differently.
Publication 4 showed that high temperature triggered the gelation of NT2RepCT condensate in E. coli., which enhanced the partitioning of drug molecules in gelated condensates. This recapitulated a key feature of gelated NT2RepCT condensates induced by laser in vitro conditions.
Publication 5 explored different protein design strategies for engineering adhesive proteins into synthetic condensates. The trend of how condensate properties change with different strategies was similar between E. coli and purified systems, although the exact properties can differ. Notably, adding a folded dimerizing domain to the disordered adhesive protein sequences can enhance condensate formation in cells and invitro, and improve underwater adhesion.
Description
Supervising professor
Linder, Markus, Prof., Aalto University, Department of Bioproducts and Biosystems, FinlandThesis advisor
Linder, Markus, Prof., Aalto University, Department of Bioproducts and Biosystems, FinlandOther note
Parts
- [Publication 1]: Feng, Jianhui; Osmekhina, Ekaterina; Timonen, Jaakko V.I.; Linder, Markus B. 2025. Effects of Sup35 overexpression on the formation, morphology, and physiological functions of intracellular Sup35 assemblies. American Society for Microbiology. Applied and Environmental Microbiology, volume 91, issue 3, pages 3050-3063. ISSN 1098-5336.
Full text in Acris/Aaltodoc: https://urn.fi/URN:NBN:fi:aalto-202505073515DOI: 10.1128/aem.01703-24 View at publisher
- [Publication 2]: Feng, Jianhui; Gabryelczyk, Bartosz; Tunn, Isabell; Osmekhina, Ekaterina; Linder, Markus B.. 2023. A Minispidroin Guides the Molecular Design for Cellular Condensation Mechanisms in S. cerevisiae. American Chemical Society. ACS Synthetic Biology, volume 12, issue 10, pages 3050-3063. ISSN 2161-5063.
Full text in Acris/Aaltodoc: https://urn.fi/URN:NBN:fi:aalto-202312117193DOI: 10.1021/acssynbio.3c00374 View at publisher
- [Publication 3]: Gabryelczyk, Bartosz; Sammalisto, Fred-Eric; Gandier, Julie-Anne; Feng, Jianhui; Beaune, Grégory; Timonen, Jaakko V.I.; Linder, Markus B. 2022. Recombinant protein condensation inside E. coli enables the development of building blocks for bioinspired materials engineering – Biomimetic spider silk protein as a case study. Elsevier Ltd. Materials Today Bio, volume 17. ISSN: 2590-0064.
Full text in Acris/Aaltodoc: https://urn.fi/URN:NBN:fi:aalto-202212076760DOI: 10.1016/j.mtbio.2022.100492 View at publisher
- [Publication 4]: Leppert, Axel; Feng, Jianhui; Railaite, Vaida; Pessatti, Tomas Bohn; Cerrato, Carmine P.; Mörman, Cecilia; Osterholz, Hannah; Lane, David P.; Maia, Filipe R. N. C.; Linder, Markus B.; Rising, Anna; Landreh, Michael; 2024. Controlling Drug Partitioning in Individual Protein Condensates through Laser-Induced Microscale Phase Transitions. American Chemical Society. Journal of the American Chemical Society, volume 146, issue 28, pages 19555–19565. ISSN: 0002-7863.
Full text in Acris/Aaltodoc: https://urn.fi/URN:NBN:fi:aalto-202408285966DOI: 10.1021/jacs.4c06688 View at publisher
- [Publication 5]: Feng, Jianhui; Aspelin, Helena; Roas-Escalona, Nelmary; Tunn, Isabell; Linder, Markus B. Initial assessment of condensate properties of engineered adhesive proteins in E. coli enables the development of underwater adhesives with strong bonding strength. (Submitted)