title: Expression and purification of active, stabilized trimethyllysine hydroxylase.
abstract: 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.
authors.affiliation: Latvian Biomedical Research and Study Centre, Ratsupites 1, Riga LV-1067, Latvia. Electronic address: andris@biomed.lu.lv.
authors.forename: Andris
authors.initials: A
authors.lastname: Kazaks
authors.affiliation: Latvian Institute of Organic Synthesis, Aizkraukles 63, Riga LV-1006, Latvia.
authors.forename: Marina
authors.initials: M
authors.lastname: Makrecka-Kuka
authors.affiliation: Latvian Institute of Organic Synthesis, Aizkraukles 63, Riga LV-1006, Latvia.
authors.forename: Janis
authors.initials: J
authors.lastname: Kuka
authors.affiliation: Latvian Biomedical Research and Study Centre, Ratsupites 1, Riga LV-1067, Latvia.
authors.forename: Tatyana
authors.initials: T
authors.lastname: Voronkova
authors.affiliation: Latvian Biomedical Research and Study Centre, Ratsupites 1, Riga LV-1067, Latvia.
authors.forename: Inara
authors.initials: I
authors.lastname: Akopjana
authors.affiliation: Latvian Institute of Organic Synthesis, Aizkraukles 63, Riga LV-1006, Latvia.
authors.forename: Solveiga
authors.initials: S
authors.lastname: Grinberga
authors.affiliation: Latvian Institute of Organic Synthesis, Aizkraukles 63, Riga LV-1006, Latvia.
authors.forename: Osvalds
authors.initials: O
authors.lastname: Pugovics
authors.affiliation: Latvian Biomedical Research and Study Centre, Ratsupites 1, Riga LV-1067, Latvia.
authors.forename: Kaspars
authors.initials: K
authors.lastname: Tars
mesh_terms: D002331:Carnitine / Q000096:biosynthesis*; D018833:Chaperonins / Q000235:genetics; D004789:Enzyme Activation; D004926:Escherichia coli; D058619:Maltose-Binding Proteins / Q000235:genetics; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000302:isolation & purification; D050563:gamma-Butyrobetaine Dioxygenase / Q000378:metabolism
chemical_list: D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins; D006899:Mixed Function Oxygenases; C025931:trimethyl-lysine hydroxylase; D050563:gamma-Butyrobetaine Dioxygenase; D018833:Chaperonins; D002331:Carnitine
keywords: Carnitine; Chaperonins GroES/EL; Maltose-binding protein; Trimethyllysine hydroxylase