دورية أكاديمية
Unravelling the Mechanism of Elastin-like Polypeptide-Enzyme Fusion Stabilization in Organic Solvents.
| العنوان: | Unravelling the Mechanism of Elastin-like Polypeptide-Enzyme Fusion Stabilization in Organic Solvents. |
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| المؤلفون: | Darji S; Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, United States.; Purdue Institute for Cancer Research, Purdue University, West Lafayette, Indiana 47907, United States., Aayush A; Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, United States.; Purdue Institute for Cancer Research, Purdue University, West Lafayette, Indiana 47907, United States., Estes KM; Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, United States., Strock JD; Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, United States., Thompson DH; Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, United States.; Purdue Institute for Cancer Research, Purdue University, West Lafayette, Indiana 47907, United States. |
| المصدر: | Biomacromolecules [Biomacromolecules] 2024 Jan 08; Vol. 25 (1), pp. 272-281. Date of Electronic Publication: 2023 Dec 20. |
| نوع المنشور: | Journal Article; Research Support, N.I.H., Extramural |
| اللغة: | English |
| بيانات الدورية: | Publisher: American Chemical Society Country of Publication: United States NLM ID: 100892849 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1526-4602 (Electronic) Linking ISSN: 15257797 NLM ISO Abbreviation: Biomacromolecules Subsets: MEDLINE |
| أسماء مطبوعة: | Original Publication: Washington, DC : American Chemical Society, c2000- |
| مواضيع طبية MeSH: | Elastin-Like Polypeptides* , Micelles*, Peptides/chemistry ; Elastin/chemistry ; Solvents ; Recombinant Fusion Proteins |
| مستخلص: | Elastin-like polypeptides (ELP) are a class of materials that are widely used as purification tags and in potential therapeutic applications. We have used the hydrophobic nature of ELP to extract them into organic solvents and precipitate them to obtain highly pure materials. Although many different types of ELP have been rapidly purified in this manner, the underlying mechanism for this process and its ability to retain functional proteins within organic phase-rich media has been unclear. A cleavable ELP-Intein construct fused with the enzyme chorismate mutase (ELP-I-Cm2) was used to better understand the organic solvent extraction process for ELP and the factors impacting the retention of enzyme activity. Our extraction studies indicated that a cell lysis step was essential to stabilize the ELP-I-Cm2 in the organic phase, prevent intein cleavage, and extract the fusion protein with high efficiency and retained activity. Circular dichroism and infrared spectroscopic characterization of ELP-I-Cm2 in organic solvents and aqueous solutions of the extracted and precipitated material indicated that the ELP secondary structure was retained in both environments. Atomic force microscopy and negative stain transmission electron microscopy imaging of ELP-I-Cm2 in organic solvents revealed highly regular circular features that were ∼50 nm in diameter, in contrast to larger (>100 nm) irregular features found in aqueous solutions. Since reverse micelles have often been used in catalytic processes, we evaluated the enzymatic activity of the ELP-I-Cm2 reversed micelles in different organic solvent mixtures and found that Cm2-mediated reactions in organic media were of comparable rate and efficiency to those in aqueous media. Based on these findings, we report an exciting new opportunity for ELP-enzyme fusion applications by exploiting their ability to form catalytically active reverse micelles in organic media. |
| معلومات مُعتمدة: | L30 CA231680 United States CA NCI NIH HHS |
| المشرفين على المادة: | 0 (Elastin-Like Polypeptides) 0 (Micelles) 0 (Peptides) 9007-58-3 (Elastin) 0 (Solvents) 0 (Recombinant Fusion Proteins) |
| تواريخ الأحداث: | Date Created: 20231220 Date Completed: 20240109 Latest Revision: 20240208 |
| رمز التحديث: | 20240209 |
| DOI: | 10.1021/acs.biomac.3c00982 |
| PMID: | 38118170 |
| قاعدة البيانات: | MEDLINE |
Find this issue from the American Chemical Society | تدمد: | 1526-4602 |
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| DOI: | 10.1021/acs.biomac.3c00982 |