CRD generated by pCARGHO: A new efficient lectin-based affinity tag method for safe, simple and low-cost protein purification.

Fiche publication

Date publication

octobre 2018

Journal

Biotechnology journal

Auteurs

Membres identifiés du Cancéropôle Est :
Pr BOSCHI-MULLER Sandrine , Pr JOUZEAU Jean-Yves

Tous les auteurs :
Kriznik A, Yéléhé-Okouma M, Lec JC, Groshenry G, Le Cordier H, Charron C, Quinternet M, Mazon H, Talfournier F, Boschi-Muller S, Jouzeau JY, Reboul P

Lien Pubmed

https://www.ncbi.nlm.nih.gov/pubmed/30298550

Résumé

Purification of recombinant proteins remains a bottleneck for downstream processing. We engineered a new galectin 3 truncated form (CRD ), functionally and structurally characterized, with preserved solubility and lectinic activity. Taking advantage of these properties, we designed an expression vector (pCARGHO), suitable for CRD -tagged protein expression in prokaryotes. CRD 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), CRD and TEV are efficiently eliminated using cationic exchange chromatography. When pI of the protein of interest and CRD were close, other chromatographic methods were successfully tested. Using CRD tag, we purified several proteins from prokaryote and eukaryote origin and demonstrated as examples, the preservation of both Escherichia coli Thioredoxin 1 and human CDC25B 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.