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Expression and characterisation of human glycerol kinase: the role of solubilising agents and molecular chaperones.
Rani, Riva Mary; Syngkli, Superior; Nongkhlaw, Joplin; Das, Bidyadhar.
Afiliación
  • Rani RM; Biological Chemistry Laboratory, Department of Zoology, North-Eastern Hill University, Shillong 793022, India.
  • Syngkli S; Biological Chemistry Laboratory, Department of Zoology, North-Eastern Hill University, Shillong 793022, India.
  • Nongkhlaw J; Biological Chemistry Laboratory, Department of Zoology, North-Eastern Hill University, Shillong 793022, India.
  • Das B; Biological Chemistry Laboratory, Department of Zoology, North-Eastern Hill University, Shillong 793022, India.
Biosci Rep ; 43(4)2023 04 21.
Article en En | MEDLINE | ID: mdl-37021775
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.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Diabetes Mellitus Tipo 2 / Glicerol Quinasa Límite: Humans Idioma: En Revista: Biosci Rep Año: 2023 Tipo del documento: Article País de afiliación: India Pais de publicación: Reino Unido
Texto completo
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XML
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Diabetes Mellitus Tipo 2 / Glicerol Quinasa Límite: Humans Idioma: En Revista: Biosci Rep Año: 2023 Tipo del documento: Article País de afiliación: India Pais de publicación: Reino Unido