Expression and characterisation of human glycerol kinase: the role of solubilising agents and molecular chaperones.
Type 2 diabetes mellitus
glycerol kinase
molecular chaperones
sarkosyl
solubilising agents
Journal
Bioscience reports
ISSN: 1573-4935
Titre abrégé: Biosci Rep
Pays: England
ID NLM: 8102797
Informations de publication
Date de publication:
21 04 2023
21 04 2023
Historique:
received:
08
11
2022
revised:
11
03
2023
accepted:
06
04
2023
medline:
24
4
2023
pubmed:
7
4
2023
entrez:
6
4
2023
Statut:
ppublish
Résumé
Glycerol kinase (GK; EC 2.7.1.30) facilitates the entry of glycerol into pathways of glucose and triglyceride metabolism and may play a potential role in Type 2 diabetes mellitus (T2DM). However, the detailed regulatory mechanisms and structure of the human GK are unknown. The human GK gene was cloned into the pET-24a(+) vector and over-expressed in Escherichia coli BL21 (DE3). Since the protein was expressed as inclusion bodies (IBs), various culture parameters and solubilising agents were used but they did not produce bioactive His-GK; however, co-expression of His-GK with molecular chaperones, specifically pKJE7, achieved expression of bioactive His-GK. The overexpressed bioactive His-GK was purified using coloumn chromatography and characterised using enzyme kinetics. The overexpressed bioactive His-GK was purified apparently to homogeneity (∼295-fold) and characterised. The native His-GK was a dimer with a monomeric molecular weight of ∼55 kDa. Optimal enzyme activity was observed in TEA buffer (50 mM) at 7.5 pH. K+ (40 mM) and Mg2+ (2.0 mM) emerged as prefered metal ions for His-GK activity with specific activity 0.780 U/mg protein. The purified His-GK obeyed standard Michaelis-Menten kinetics with Km value of 5.022 µM (R2=0.927) for its substrate glycerol; whereas, that for ATP and PEP was 0.767 mM (R2=0.928) and 0.223 mM (R2=0.967), respectively. Other optimal parameters for the substrate and co-factors were also determined. The present study demonstrates that co-expression of molecular chaperones assists with the expression of bioactive human GK for its characterisation.
Sections du résumé
BACKGROUND
Glycerol kinase (GK; EC 2.7.1.30) facilitates the entry of glycerol into pathways of glucose and triglyceride metabolism and may play a potential role in Type 2 diabetes mellitus (T2DM). However, the detailed regulatory mechanisms and structure of the human GK are unknown.
METHODS
The human GK gene was cloned into the pET-24a(+) vector and over-expressed in Escherichia coli BL21 (DE3). Since the protein was expressed as inclusion bodies (IBs), various culture parameters and solubilising agents were used but they did not produce bioactive His-GK; however, co-expression of His-GK with molecular chaperones, specifically pKJE7, achieved expression of bioactive His-GK. The overexpressed bioactive His-GK was purified using coloumn chromatography and characterised using enzyme kinetics.
RESULTS
The overexpressed bioactive His-GK was purified apparently to homogeneity (∼295-fold) and characterised. The native His-GK was a dimer with a monomeric molecular weight of ∼55 kDa. Optimal enzyme activity was observed in TEA buffer (50 mM) at 7.5 pH. K+ (40 mM) and Mg2+ (2.0 mM) emerged as prefered metal ions for His-GK activity with specific activity 0.780 U/mg protein. The purified His-GK obeyed standard Michaelis-Menten kinetics with Km value of 5.022 µM (R2=0.927) for its substrate glycerol; whereas, that for ATP and PEP was 0.767 mM (R2=0.928) and 0.223 mM (R2=0.967), respectively. Other optimal parameters for the substrate and co-factors were also determined.
CONCLUSION
The present study demonstrates that co-expression of molecular chaperones assists with the expression of bioactive human GK for its characterisation.
Identifiants
pubmed: 37021775
pii: 232867
doi: 10.1042/BSR20222258
pmc: PMC10130975
pii:
doi:
Substances chimiques
Glycerol Kinase
EC 2.7.1.30
Glycerol
PDC6A3C0OX
Molecular Chaperones
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Commentaires et corrections
Type : ErratumIn
Informations de copyright
© 2023 The Author(s).
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