Anaerobic Expression and Purification of Holo-CCIS, an Artificial Iron-sulfur Protein.
| Autor: | Jagilinki BP; Migal-Galilee Research Institute, Kiryat Shmona 11016, Israel.; Center for Advanced Biotechnology and Medicine and the Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, Rutgers University, Piscataway, New Jersey 08854, USA., Paluy I; Migal-Galilee Research Institute, Kiryat Shmona 11016, Israel., Nanda V; Center for Advanced Biotechnology and Medicine and the Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, Rutgers University, Piscataway, New Jersey 08854, USA., Noy D; Migal-Galilee Research Institute, Kiryat Shmona 11016, Israel. |
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| Jazyk: | angličtina |
| Zdroj: | Bio-protocol [Bio Protoc] 2021 Sep 20; Vol. 11 (18), pp. e4169. Date of Electronic Publication: 2021 Sep 20 (Print Publication: 2021). |
| DOI: | 10.21769/BioProtoc.4169 |
| Abstrakt: | Iron-sulfur proteins are ubiquitous among all living organisms and are indispensable for almost all metabolic pathways ranging from photosynthesis, respiration, nitrogen, and carbon dioxide cycles. The iron-sulfur clusters primarily serve as electron acceptors and donors and transfer electrons to active sites of various enzymes, thus driving the energy metabolism. Prokaryotes like E. coli have ISC and SUF pathways that help in the assembly and maturation of iron-sulfur proteins. These iron-sulfur proteins, especially with [4Fe-4S] clusters, are highly sensitive to molecular oxygen, and it would be advantageous if the de novo proteins and native proteins having iron-sulfur binding sites are expressed and isolated under anaerobic conditions. Bacterially assembled iron-sulfur proteins, when isolated and purified anaerobically, exhibit improved biochemical and biophysical stabilities in comparison to the counterparts expressed and purified aerobically and reconstituted under anaerobic conditions. This protocol outlines the expression and purification of the artificial protein, Coiled-Coil Iron-Sulfur (CCIS). It may be deployed to both natural and artificial [4Fe-4S] proteins when heterologously expressed in E. coli . Competing Interests: Competing interestsThe authors declare no competing interests. (Copyright © 2021 The Authors; exclusive licensee Bio-protocol LLC.) |
| Databáze: | MEDLINE |
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