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Efficient new lectin-based affinity tag allowing simple, safe and low-cost recombinant protein purification

Abstract : Even today, the purification of recombinant proteins remains a bottleneck for downstream research. In order to carry out fundamental studies on the Carbohydrate Recognition Domain (CRD) of the human galectin 3, we developed a new truncated form (CRDSAT), functionally (iTC) and structurally (X-ray, native MS) characterized and whose solubility and lectinic activity were preserved. By taking advantage of these properties, we decided to develop and patent, a single step purification method for CRDSAT-tagged fusion proteins1. To this aim, we designed an expression vector (pCARGHO), suitable for protein expression in prokaryotes. The expressed tag (CRDSAT) binds to lactose-Sepharose with a high specificity and facilitates the solubilisation of fusion proteins2. This tag is structurally stable even in reducing conditions and, due to its small size (18.7 kDa), can be easily cleaved from fusion proteins, by tobacco etch virus (TEV) protease. CRDSAT and TEV can be efficiently eliminated by cationic exchange chromatography, as their pI is basic. Furthermore, other chromatographic methods were successfully tested when the pI of the protein of interest is close to the CRDSAT’ one. By using this technique, we purified several proteins from prokaryote and eukaryote origin and demonstrated its efficiency. Globally, yields of pure proteins obtained after tag removal were about 5 to 20 mg/L of bacterial culture. Our purification method displays various advantages that may be of great interest for academic laboratories, biotechnology and pharmaceutical companies. Contact: Société d'Accélération du Transfert de Technologie GRAND EST daniel.kirchherr@sattge.fr ; aude.hyardin@sattge.fr References: 1 Patent WO2017194888; 2Carrasco et al., Cellular and Molecular Immunology 2018, accepted.
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https://hal.univ-lorraine.fr/hal-01719814
Contributor : Pascal Reboul <>
Submitted on : Wednesday, February 28, 2018 - 3:00:49 PM
Last modification on : Friday, September 24, 2021 - 9:36:03 AM

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  • HAL Id : hal-01719814, version 1

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Alexandre Kriznik, Melissa Yelehe-Okouma, Christophe Charron, Hortense Mazon, Hélène Le Cordier, et al.. Efficient new lectin-based affinity tag allowing simple, safe and low-cost recombinant protein purification. The 14th International Symposium on BioChromatography and Nanoseparations, May 2018, Bordeaux, France. ⟨hal-01719814⟩

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