Overexpression and purification of U24 from human herpesvirus type-6 in E. coli: unconventional use of oxidizing environments with a maltose binding protein-hexahistine dual tag to enhance membrane protein yield
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Notice bibliographique
Résumé
BACKGROUND: Obtaining membrane proteins in sufficient quantity for biophysical study and biotechnological applications has been a difficult task. Use of the maltose binding protein/hexahistidine dual tag system with E.coli as an expression host is emerging as a high throughput method to enhance membrane protein yield, solubility, and purity, but fails to be effective for certain proteins. Optimizing the variables in this system to fine-tune for efficiency can ultimately be a daunting task. To identify factors critical to success in this expression system, we have selected to study U24, a novel membrane protein from Human Herpesvirus type-6 with potent immunosuppressive ability and a possible role in the pathogenesis of the disease multiple sclerosis. RESULTS: We expressed full-length U24 as a C-terminal fusion to a maltose binding protein/hexahistidine tag and examined the effects of temperature, growth medium type, cell strain type, oxidizing vs. reducing conditions and periplasmic vs. cytoplasmic expression location. Temperature appeared to have the greatest effect on yield; at 37°C full-length protein was either poorly expressed (periplasm) or degraded (cytoplasm) whereas at 18°C, expression was improved especially in the periplasm of C41(DE3) cells and in the cytoplasm of oxidizing Δtrx/Δgor mutant strains, Origami 2 and SHuffle. Expression of the fusion protein in these strains were estimated to be 3.2, 5.3 and 4.3 times greater, respectively, compared to commonly-used BL21(DE3) cells. We found that U24 is isolated with an intramolecular disulfide bond under these conditions, and we probed whether this disulfide bond was critical to high yield expression of full-length protein. Expression analysis of a C21SC37S cysteine-free mutant U24 demonstrated that this disulfide was not critical for full-length protein expression, but it is more likely that strained metabolic conditions favour factors which promote protein expression. This hypothesis is supported by the fact that use of minimal media could enhance protein production compared to nutrient-rich LB media. CONCLUSIONS: We have found optimal conditions for heterologous expression of U24 from Human Herpesvirus type-6 in E.coli and have demonstrated that milligram quantities of pure protein can be obtained. Strained metabolic conditions such as low temperature, minimal media and an oxidizing environment appeared essential for high-level, full-length protein production and this information may be useful for expressing other membrane proteins of interest.
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Prédiction distillée sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Apprise à partir de 10 348 étiquettes directes de Codex et de 10 348 étiquettes directes de Gemma. Le mode candidate est l'union des têtes enseignantes seuillées; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont ni des étiquettes humaines ni des étiquettes directes de modèles de pointe.
Scores Codex et Gemma par catégorie
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,000 | 0,000 |
| Méta-épidémiologie (sens strict) | 0,000 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,000 |
| Bibliométrie | 0,000 | 0,000 |
| Études des sciences et des technologies | 0,000 | 0,000 |
| Communication savante | 0,000 | 0,000 |
| Science ouverte | 0,000 | 0,000 |
| Intégrité de la recherche | 0,000 | 0,000 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,000 | 0,000 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découle