POMPOMS: Crosslinked biomolecular condensates as a versatile platform for multifunctional protein microparticles
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Abstract
ABSTRACT Protein-based microparticles are promising materials for applications such as biocatalysis and biomolecular capture, yet their fabrication by existing techniques remains challenging due to protein denaturation or lack of spatial control. Here, we present a method for synthesizing microscale protein-based materials by chemically crosslinking biomolecular condensates. Leveraging the liquid-liquid phase separation behavior of intrinsically disordered RGG domains, we sequestered RGG-tagged fusion proteins into droplets, then we solidified them into porous microparticles using the homobifunctional, amine-reactive crosslinker BS³. By modulating protein concentration and condensate coalescence, we controlled microparticle size from 40 µm. We then demonstrated three encodable functionalities: We used the SpyCatcher/SpyTag system to capture cargo proteins, we crosslinked core-shell condensates to generate microparticles with controlled spatial organization, and we immobilized a thermostable alcohol dehydrogenase with 31% retained enzymatic activity. These POMPOMS (protein-based, self-organized microparticles of multifunctional significance) represent a sustainable, tunable platform for versatile protein-based materials.
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ABSTRACT
Protein-based microparticles are promising materials for applications such as biocatalysis and biomolecular capture, yet their fabrication by existing techniques remains challenging due to protein denaturation or lack of spatial control. Here, we present a method for synthesizing microscale protein-based materials by chemically crosslinking biomolecular condensates. Leveraging the liquid-liquid phase separation behavior of intrinsically disordered RGG domains, we sequestered RGG-tagged fusion proteins into droplets, then we solidified them into porous microparticles using the homobifunctional, amine-reactive crosslinker BS³. By modulating protein concentration and condensate coalescence, we controlled microparticle size from 40 µm. We then demonstrated three encodable functionalities: We used the SpyCatcher/SpyTag system to capture cargo proteins, we crosslinked core-shell condensates to generate microparticles with controlled spatial organization, and we immobilized a thermostable alcohol dehydrogenase with 31% retained enzymatic activity. These POMPOMS (protein-based, self-organized microparticles of multifunctional significance) represent a sustainable, tunable platform for versatile protein-based materials.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
New experiments exploring the effects of crosslinker concentration on microparticle properties. New figure 3 added. Supplemental files added.
ABBREVIATIONS
- ANOVA
- analysis of variance
- AU
- arbitrary units
- BCA
- bicinchoninic acid
- BS3
- bis(sulfosuccinimidyl) suberate
- BsADH
- alcohol dehydrogenase from Geobacillus stearothermophilus
- CLEA
- crosslinked enzyme aggregate.
- CV
- column volume
- DNA
- deoxyribonucleoic acid
- EDTA
- ethylenediaminetetraacetic acid
- FPLC
- fast protein liquid chromatography
- FRAP
- fluorescence recovery after photobleaching
- GFP
- green fluorescent protein
- IDP
- intrinsically disordered protein
- IPTG
- iso-propyl-ß-D-1-thiogalactopyranoside
- kDa
- kilodalton
- LB
- lysogeny broth
- LLPS
- liquid-liquid phase separation
- MBP
- maltose-binding protein
- MES
- 2-morpholinoethanesulfonic acid
- MW
- molecular weight
- MWCO
- molecular weight cutoff
- NA
- numerical aperture
- NAD+
- nicotinamide adenine dinucleotide (oxidized)
- NADH
- nicotinamide adenine dinucleotide (reduced)
- NIR
- near infrared
- OD
- optical density
- PEG
- polyethylene glycol
- POMPOMS
- protein-based, self-organized micro-particles of multifunctional significance
- PS
- polystyrene
- Rh
- hydrodynamic radius
- SDS
- sodium dodecyl sulfate
- SDS-PAGE
- sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- SEM
- standard error of means
- TB
- Terrific Broth
- Tm’
- apparent melting temperature
- TRITC
- tetramethylrhodamine isothiocyanate
- UCST
- upper critical solution temperature
- UV
- ultra-violet
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- last seen: 2026-05-20T01:45:00.602351+00:00
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- last seen: 2026-06-13T06:42:57.164913+00:00