Expression and purification of active, stabilized trimethyllysine hydroxylase

•TMLH expressed in E. coli either alone, or as MBP fusion is mainly insoluble.•Co-expression of MBP–TMLH with chaperonins GroES/EL markedly improves solubility.•Purified MBP–TMLH is stable and functionally active. Trimethyllysine hydroxylase (TMLH) catalyses the first step in carnitine biosynthesis...

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Vydáno v:Protein expression and purification Ročník 104; s. 1 - 6
Hlavní autoři: Kazaks, Andris, Makrecka-Kuka, Marina, Kuka, Janis, Voronkova, Tatyana, Akopjana, Inara, Grinberga, Solveiga, Pugovics, Osvalds, Tars, Kaspars
Médium: Journal Article
Jazyk:angličtina
Vydáno: United States Elsevier Inc 01.12.2014
Témata:
Carnitine
Carnitine - biosynthesis
Chaperonins - genetics
Chaperonins GroES/EL
Enzyme Activation
Escherichia coli
gamma-Butyrobetaine Dioxygenase - metabolism
Maltose-binding protein
Maltose-Binding Proteins - genetics
Mixed Function Oxygenases - genetics
Mixed Function Oxygenases - isolation & purification
Recombinant Fusion Proteins - genetics
Recombinant Fusion Proteins - isolation & purification
Trimethyllysine hydroxylase
Chaperonins GroES/EL
Maltose-binding protein
Trimethyllysine hydroxylase
Carnitine
ISSN:1046-5928, 1096-0279, 1096-0279
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Shrnutí:•TMLH expressed in E. coli either alone, or as MBP fusion is mainly insoluble.•Co-expression of MBP–TMLH with chaperonins GroES/EL markedly improves solubility.•Purified MBP–TMLH is stable and functionally active. Trimethyllysine hydroxylase (TMLH) catalyses the first step in carnitine biosynthesis – the conversion of N6,N6,N6-trimethyl-l-lysine to 3-hydroxy-N6,N6,N6-trimethyl-l-lysine. By changing carnitine availability it is possible to optimise cardiac energy metabolism, that is beneficial under certain ischemic conditions. Previous efforts have been devoted towards the inhibition of gamma-butyrobetaine dioxygenase, which catalyses the last step in carnitine biosynthesis. However, the effects of TMLH activity regulation are currently unexplored. To facilitate the development of specific ligands of TMLH, large quantities of recombinant protein are necessary for downstream binding and structural studies. Here, we describe an efficient system for expressing and purifying active and stable TMLH as a maltose-binding protein fusion in Escherichiacoli.
Bibliografie:ObjectType-Article-1
SourceType-Scholarly Journals-1
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content type line 23
ISSN:1046-5928
1096-0279
1096-0279
DOI:10.1016/j.pep.2014.09.002