A novel approach to the expression and purification of recombinant Xenopus Cdc25C
| Autor: | Khaled Machaca, Abdulrahman Al-Abdulmalek, Ghizlaine Bendriss, Stephanie Schaefer-Ramadan, Satanay Hubrack |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Cdc25 Xenopus Molecular Sequence Data Phosphatase CDC25C Gene lac operon Xenopus Proteins law.invention 03 medical and health sciences law Escherichia coli Animals cdc25 Phosphatases Gene family Amino Acid Sequence Cyclin-dependent kinase 1 Base Sequence biology biology.organism_classification Molecular biology Recombinant Proteins Cell biology 030104 developmental biology Recombinant DNA biology.protein Oligopeptides Biotechnology |
| Zdroj: | Protein Expression and Purification. 120:148-152 |
| ISSN: | 1046-5928 |
| DOI: | 10.1016/j.pep.2015.12.007 |
| Popis: | The Cdc25 family encodes dual specificity protein phosphatases that play critical roles in cell cycle progression. Activation of the Cdc25C represents a primary driver for meiosis progression in Xenopus oocytes. Given its central role in meiosis the Xenopus Cdc25C has been studied extensively, however purification of the recombinant protein is difficult thus preventing better characterization of its function. Here we describe methods to overcome these difficulties resulting in the production of high purity and yield recombinant Xenopus Cdc25C. We use a synthetic Xenopus Cdc25C gene that was codon optimized for expression in E. coli. We further combine an N-terminal His-tag with a C-terminal Strep-tag II, to isolate extremely pure full-length Cdc25C protein. The recombinant Xenopus Cdc25C is active both in vitro using a phosphatase assay and in vivo when injected into Xenopus oocytes. This new approach should be applicable to the purification of other members of the Cdc25 gene family. |
| Databáze: | OpenAIRE |
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