High-yield Production of Recombinant Platelet Factor 4 Protein by Harnessing and Honing the Gram-negative Bacterial Secretory Apparatus
Notice bibliographique
Résumé
Abstract Background Platelet factor 4 is a cytokine released into the bloodstream by activated platelets and plays a pivotal role in heparin-induced thrombocytopenia etiology and diagnosis. Therefore, a sustainable source of recombinant PF4 with structural and functional similarity to its native form is urgently needed to be used in diagnostic procedures. To this end, a three-in-one primary construct was designed and custom synthesized based on the pET26b backbone from which three secondary constructs could be derived each capable of employing either type I, type II secretory or cytoplasmic pathways. Protein expression and secretion were performed in Escherichia coli BL-21 (DE3) and were confirmed by SDS-PAGE and Western blotting. To further enhance protein secretion, the effect of several controllable factors including IPTG, Triton X-100, Sucrose, and Glycine were individually investigated at first. In the next step, according to fractional factorial approach, the synergistic effect of IPTG, Triton X-100, and Glycine on secretion was further investigated. To ascertain the structure and function of the secreted recombinant proteins, Dynamic light scattering was utilized and confirmed rPF4 tetramerization and heparin-mediated ultra-large complex formation. Moreover, Raman spectroscopy was exploited to determine the rPF4 secondary structure. Results Type II secretory pathway was proven to be superior over type I in case of rPF4 secretion into the extracellular milieu. Protein secretion mediated by Type II was enhanced to approximately more than 700 μg/ml. Large quantities of native rPF4 up to 20 mg was purified upon a minor scale up to 40 ml of culture medium. Dynamic light scattering unveiled native rPF4 quaternary structure revealing the formation of tetramers having an average size of 10 nm and formation of larger complexes of approximately 100-1200 nm in size following heparin supplementation, implying proper protein folding, tetramerization, and antigenicity. Analysis of the Zeta potential on approximately 600 μg/ml of rPF4 revealed a 98 mV positive charge which further confirms protein folding. Moreover, rPF4 secondary structure was determined to be 43.5% Random coil, 32.5% β-sheet, 18.6 % α-helix and 4.9 % Turn, which is in perfect agreement with the native structure. Conclusion our results indicate that the gram-negative type II bacterial secretory system holds a great promise to be employed as a reliable protein production strategy with favorable industrial application. However, further efforts are required to realize the full potential of secretory pathways regarding their application to proteins with distinct characteristics. Abstract Figure Graphical Abstract. rPF4 secretion mediated by type 2 secretory system. The pelB signal sequence directs protein export into the extracellular milieu through the SecYEG translocon complex in a process assisted by SecB chaperone. A) Indicates protein secretion before supplementation with additives and B) indicates secretion in the presence of additives.
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Comment cette classification a été obtenuedéplier
Prédiction machine sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Le volet Gemma est une étiquette directe du modèle pour chaque travail de la base, lue sur la notice réduite au titre. Le volet Codex est un classifieur appris des 10 348 étiquettes directes de Codex et calibré sur les taux pondérés de l'échantillon; les champs sans appui suffisant ne portent aucun appel Codex. Le mode candidate est l'union des deux volets; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont pas des étiquettes humaines.
Scores du classifieur distillé par catégorie (deux têtes)
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,000 | 0,000 |
| Méta-épidémiologie (sens strict) | 0,001 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,001 |
| Bibliométrie | 0,000 | 0,000 |
| Études des sciences et des technologies | 0,000 | 0,000 |
| Communication savante | 0,001 | 0,000 |
| Science ouverte | 0,000 | 0,000 |
| Intégrité de la recherche | 0,000 | 0,001 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,001 | 0,001 |
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 découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule source (Gemma direct ou Codex distillé), pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».