Yield of Protein Crystallization from Metastable Liquid–Liquid Phase Separation
Preparative protein crystallization is regarded as an economically sustainable protein purification alternative to chromatography in biotechnological downstream processing. However, protein crystallization is a not-well-understood process that is usually slow and poorly reproducible. A promising str...
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2025-05-01
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| author | Shamberia Thomas Joel A. Dougay Onofrio Annunziata |
| author_facet | Shamberia Thomas Joel A. Dougay Onofrio Annunziata |
| author_sort | Shamberia Thomas |
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| description | Preparative protein crystallization is regarded as an economically sustainable protein purification alternative to chromatography in biotechnological downstream processing. However, protein crystallization is a not-well-understood process that is usually slow and poorly reproducible. A promising strategy for enhancing protein crystallization is exploiting the metastable liquid–liquid phase separation (LLPS) of protein solutions. Here, we report an enhancement of lysozyme-crystallization yield by using a combination of two additives under LLPS conditions. The first additive, NaCl (0.15 M), is necessary to introduce protein–protein attractive interactions and induce LLPS by lowering temperature. The second additive, 4-(2-hydroxyethyl)-1-piperazineethanesulfonate (HEPES, 0.10 M, pH 7.4), accumulates in the metastable protein-rich liquid phase and thermodynamically stabilizes lysozyme crystals. We found that this combination of additives leads to crystallization yields of higher than 90% under LLPS conditions at a lysozyme concentration of 5% by weight and a fairly low ionic strength (0.2 M) within an operational time of the order of one hour. This crystallization yield is more than three-fold larger than that obtained from samples containing NaCl without HEPES at the same pH and ionic strength. Moreover, we determined crystallization yield as a function of incubation time, and temperature below and above the LLPS boundary. As crystallization temperature intersects with LLPS temperature, a significant increase in crystallization yield is observed. This is consistent with LLPS boosting protein crystallization. Our work suggests a possible strategy for increasing the crystallization success of other proteins, with applications in protein purification. |
| format | Article |
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| issn | 1420-3049 |
| language | English |
| publishDate | 2025-05-01 |
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| spelling | doaj-art-394aeb2d8689421ba03a4664dfcc8bcf2025-08-20T02:33:06ZengMDPI AGMolecules1420-30492025-05-013011237110.3390/molecules30112371Yield of Protein Crystallization from Metastable Liquid–Liquid Phase SeparationShamberia Thomas0Joel A. Dougay1Onofrio Annunziata2Department of Chemistry and Biochemistry, Texas Christian University, Fort Worth, TX 76109, USADepartment of Chemistry and Biochemistry, Texas Christian University, Fort Worth, TX 76109, USADepartment of Chemistry and Biochemistry, Texas Christian University, Fort Worth, TX 76109, USAPreparative protein crystallization is regarded as an economically sustainable protein purification alternative to chromatography in biotechnological downstream processing. However, protein crystallization is a not-well-understood process that is usually slow and poorly reproducible. A promising strategy for enhancing protein crystallization is exploiting the metastable liquid–liquid phase separation (LLPS) of protein solutions. Here, we report an enhancement of lysozyme-crystallization yield by using a combination of two additives under LLPS conditions. The first additive, NaCl (0.15 M), is necessary to introduce protein–protein attractive interactions and induce LLPS by lowering temperature. The second additive, 4-(2-hydroxyethyl)-1-piperazineethanesulfonate (HEPES, 0.10 M, pH 7.4), accumulates in the metastable protein-rich liquid phase and thermodynamically stabilizes lysozyme crystals. We found that this combination of additives leads to crystallization yields of higher than 90% under LLPS conditions at a lysozyme concentration of 5% by weight and a fairly low ionic strength (0.2 M) within an operational time of the order of one hour. This crystallization yield is more than three-fold larger than that obtained from samples containing NaCl without HEPES at the same pH and ionic strength. Moreover, we determined crystallization yield as a function of incubation time, and temperature below and above the LLPS boundary. As crystallization temperature intersects with LLPS temperature, a significant increase in crystallization yield is observed. This is consistent with LLPS boosting protein crystallization. Our work suggests a possible strategy for increasing the crystallization success of other proteins, with applications in protein purification.https://www.mdpi.com/1420-3049/30/11/2371NaClHEPESLLPS |
| spellingShingle | Shamberia Thomas Joel A. Dougay Onofrio Annunziata Yield of Protein Crystallization from Metastable Liquid–Liquid Phase Separation Molecules NaCl HEPES LLPS |
| title | Yield of Protein Crystallization from Metastable Liquid–Liquid Phase Separation |
| title_full | Yield of Protein Crystallization from Metastable Liquid–Liquid Phase Separation |
| title_fullStr | Yield of Protein Crystallization from Metastable Liquid–Liquid Phase Separation |
| title_full_unstemmed | Yield of Protein Crystallization from Metastable Liquid–Liquid Phase Separation |
| title_short | Yield of Protein Crystallization from Metastable Liquid–Liquid Phase Separation |
| title_sort | yield of protein crystallization from metastable liquid liquid phase separation |
| topic | NaCl HEPES LLPS |
| url | https://www.mdpi.com/1420-3049/30/11/2371 |
| work_keys_str_mv | AT shamberiathomas yieldofproteincrystallizationfrommetastableliquidliquidphaseseparation AT joeladougay yieldofproteincrystallizationfrommetastableliquidliquidphaseseparation AT onofrioannunziata yieldofproteincrystallizationfrommetastableliquidliquidphaseseparation |