CRD SAT generated by pCARGHO: A new efficient lectin-based affinity tag method for safe, simple and low-cost protein purification
| Autor: | Hélène Le Cordier, Guillaume Groshenry, François Talfournier, Alexandre Kriznik, Sandrine Boschi-Muller, Jean-Christophe Lec, Pascal Reboul, Jean-Yves Jouzeau, Marc Quinternet, Christophe Charron, Melissa Yelehe-Okouma, Hortense Mazon |
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| Přispěvatelé: | Ingénierie Moléculaire et Physiopathologie Articulaire (IMoPA), Université de Lorraine (UL)-Centre National de la Recherche Scientifique (CNRS), Département de Pharmacologie Clinique et Toxicologie, Centre Hospitalier Régional Universitaire, Nancy, France, Ingénierie, Biologie et Santé en Lorraine (IBSLor), Université de Lorraine (UL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Service de Pharmacologie Clinique et Toxicologie [CHRU Nancy], Centre Hospitalier Régional Universitaire de Nancy (CHRU Nancy), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lorraine (UL)-Centre National de la Recherche Scientifique (CNRS), Reboul, Pascal |
| Jazyk: | angličtina |
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
[SDV.BIO]Life Sciences [q-bio]/Biotechnology Protein purification based on new tag technology medicine.disease_cause Affinity chromatography Applied Microbiology and Biotechnology 03 medical and health sciences Protein purification [SDV.BBM] Life Sciences [q-bio]/Biochemistry Molecular Biology TEV protease medicine [SDV.BBM]Life Sciences [q-bio]/Biochemistry Molecular Biology Escherichia coli galectin-3-derived CRD-tag Expression vector Downstream processing 030102 biochemistry & molecular biology Chemistry General Medicine Fusion protein [SDV.BIO] Life Sciences [q-bio]/Biotechnology 030104 developmental biology Isoelectric point Biochemistry Molecular Medicine |
| Zdroj: | Biotechnology Journal Biotechnology Journal, Wiley-VCH Verlag, 2018, pp.e1800214. ⟨10.1002/biot.201800214⟩ Biotechnology Journal, 2019, 14 (4), pp.e1800214. ⟨10.1002/biot.201800214⟩ |
| ISSN: | 1860-6768 1860-7314 |
| DOI: | 10.1002/biot.201800214⟩ |
| Popis: | International audience; Purification of recombinant proteins remains a bottleneck for downstream processing. We engineered a new galectin 3 truncated form (CRDSAT ), functionally and structurally characterized, with preserved solubility and lectinic activity. Taking advantage of these properties, we designed an expression vector (pCARGHO), suitable for CRDSAT -tagged protein expression in prokaryotes. CRDSAT binds to lactose-Sepharose with a high specificity and facilitates solubilization of fusion proteins. This tag is structurally stable and can be easily removed from fusion proteins using TEV protease. Furthermore, due to their basic isoelectric point (pI), CRDSAT and TEV are efficiently eliminated using cationic exchange chromatography. When pI of the protein of interest and CRDSAT were close, other chromatographic methods were successfully tested. Using CRDSAT tag, we purified several proteins from prokaryote and eukaryote origin and demonstrated as examples, the preservation of both Escherichia coli Thioredoxin 1 and human CDC25Bcd activities. Overall, yields of proteins obtained after tag removal were about 5 to 50 mg per litre of bacterial culture. Our purification method displays various advantages described herein that may greatly interest academic laboratories, biotechnology and pharmaceutical companies. |
| Databáze: | OpenAIRE |
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