Effect of prepropeptide replacement on γ-carboxylation and activity of recombinant coagulation factor IX.
Coagulation Factor IX
Prepropeptide
Protein C
γ-Carboxylase
Journal
Biotechnology letters
ISSN: 1573-6776
Titre abrégé: Biotechnol Lett
Pays: Netherlands
ID NLM: 8008051
Informations de publication
Date de publication:
Aug 2022
Aug 2022
Historique:
received:
04
04
2022
accepted:
31
05
2022
pubmed:
23
6
2022
medline:
10
8
2022
entrez:
22
6
2022
Statut:
ppublish
Résumé
Based on observations indicating that the γ-carboxylase enzyme has a lower affinity for the protein C (PC) propeptide and that the γ-carboxylase region in the PC propeptide has a higher net charge, expression of recombinant chimeric factor IX (FIX) equipped with the PC propeptide was studied. The prepropeptide of FIX was replaced with that of PC by SOEing PCR and after cloning, recombinant pMT-prepro PC/FIX was transfected into insect Drosophila S2 cells. The expression and activity of expressed FIX were analyzed employing antigen and activity analyses 72 h of post-induction with copper. Higher secretion (1.2 fold) and activity (1.6 fold) levels were observed for chimeric prepro- PC/FIX in relation to wild-type FIX. Furthermore, after barium citrate precipitation, the evaluation of fully γ-carboxylated FIX indicated that more than 51% of the total FIX produced with the PC prepropeptide was fully γ-carboxylated, representing a substantial improvement (twofold) over a system employing the native FIX propeptide in which 25% of the protein is fully γ-carboxylated. The data illustrated that the expression of FIX using the PC propeptide led to much higher fully γ-carboxylated material, which is preferred to FIX constructs tolerating the sequence for the native FIX propeptide expressed in heterologous S2 systems.
Identifiants
pubmed: 35731352
doi: 10.1007/s10529-022-03269-4
pii: 10.1007/s10529-022-03269-4
doi:
Substances chimiques
Recombinant Proteins
0
Factor IX
9001-28-9
Carbon-Carbon Ligases
EC 6.4.-
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
975-984Subventions
Organisme : Hakim Sabzevari University
ID : 200
Informations de copyright
© 2022. The Author(s), under exclusive licence to Springer Nature B.V.
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