Expression and purification of untagged and histidine-tagged folate-dependent tRNA:m5U54 methyltransferase from Bacillus subtilis

Djemel Hamdane; Stèphane Skouloubris; Hannu Myllykallio; Bèatrice Golinelli-Pimpaneau

doi:10.1016/j.pep.2010.04.013

Expression and purification of untagged and histidine-tagged folate-dependent tRNA:m⁵U54 methyltransferase from Bacillus subtilis

Djemel Hamdane
, Stèphane Skouloubris
, Hannu Myllykallio
, Bèatrice Golinelli-Pimpaneau

CNRS-UPR U. Propre de Recherche 9063
Université Paris-Saclay

Résultats de recherche: Contribution à un journal › Article › Revue par des pairs

Résumé

Folate-dependent tRNA m⁵U methyltransferase TrmFO is a flavoprotein that catalyzes the C⁵-methylation of uridine at position 54 in the TψC loop of tRNA in several bacteria. Here we report the cloning and optimization of expression in Escherichia coli BL21 (DE3) of untagged, N-terminus, C-terminus (His)₆-tagged TrmFO from Bacillus subtilis. Tagged and untagged TrmFO were purified to homogeneity by metal affinity or ion exchange and heparin affinity, respectively, followed by size-exclusion chromatography. The tag did not significantly alter the expression level, flavin content, activity and secondary structure of the protein.

langue originale	Anglais
Pages (de - à)	83-89
Nombre de pages	7
journal	Protein Expression and Purification
Volume	73
Numéro de publication	1
Les DOIs	https://doi.org/10.1016/j.pep.2010.04.013
état	Publié - 1 janv. 2010

Accès au document

10.1016/j.pep.2010.04.013

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@article{8d3bcaabfa2d43f8ab49bd8eba1ec555,

title = "Expression and purification of untagged and histidine-tagged folate-dependent tRNA:m5U54 methyltransferase from Bacillus subtilis",

abstract = "Folate-dependent tRNA m5U methyltransferase TrmFO is a flavoprotein that catalyzes the C5-methylation of uridine at position 54 in the TψC loop of tRNA in several bacteria. Here we report the cloning and optimization of expression in Escherichia coli BL21 (DE3) of untagged, N-terminus, C-terminus (His)6-tagged TrmFO from Bacillus subtilis. Tagged and untagged TrmFO were purified to homogeneity by metal affinity or ion exchange and heparin affinity, respectively, followed by size-exclusion chromatography. The tag did not significantly alter the expression level, flavin content, activity and secondary structure of the protein.",

keywords = "5-Methyluridine, Expression, Flavoenzyme, Histidine tag, Purification, RNA methyltransferase, Recombinant TrmFO protein",

author = "Djemel Hamdane and St{\`e}phane Skouloubris and Hannu Myllykallio and B{\`e}atrice Golinelli-Pimpaneau",

year = "2010",

month = jan,

day = "1",

doi = "10.1016/j.pep.2010.04.013",

language = "English",

volume = "73",

pages = "83--89",

journal = "Protein Expression and Purification",

issn = "1046-5928",

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Expression and purification of untagged and histidine-tagged folate-dependent tRNA:m⁵U54 methyltransferase from Bacillus subtilis. / Hamdane, Djemel; Skouloubris, Stèphane ; Myllykallio, Hannu et al.
Dans: Protein Expression and Purification, Vol 73, Numéro 1, 01.01.2010, p. 83-89.

Résultats de recherche: Contribution à un journal › Article › Revue par des pairs

TY - JOUR

T1 - Expression and purification of untagged and histidine-tagged folate-dependent tRNA:m5U54 methyltransferase from Bacillus subtilis

AU - Hamdane, Djemel

AU - Skouloubris, Stèphane

AU - Myllykallio, Hannu

AU - Golinelli-Pimpaneau, Bèatrice

PY - 2010/1/1

Y1 - 2010/1/1

N2 - Folate-dependent tRNA m5U methyltransferase TrmFO is a flavoprotein that catalyzes the C5-methylation of uridine at position 54 in the TψC loop of tRNA in several bacteria. Here we report the cloning and optimization of expression in Escherichia coli BL21 (DE3) of untagged, N-terminus, C-terminus (His)6-tagged TrmFO from Bacillus subtilis. Tagged and untagged TrmFO were purified to homogeneity by metal affinity or ion exchange and heparin affinity, respectively, followed by size-exclusion chromatography. The tag did not significantly alter the expression level, flavin content, activity and secondary structure of the protein.

AB - Folate-dependent tRNA m5U methyltransferase TrmFO is a flavoprotein that catalyzes the C5-methylation of uridine at position 54 in the TψC loop of tRNA in several bacteria. Here we report the cloning and optimization of expression in Escherichia coli BL21 (DE3) of untagged, N-terminus, C-terminus (His)6-tagged TrmFO from Bacillus subtilis. Tagged and untagged TrmFO were purified to homogeneity by metal affinity or ion exchange and heparin affinity, respectively, followed by size-exclusion chromatography. The tag did not significantly alter the expression level, flavin content, activity and secondary structure of the protein.

KW - 5-Methyluridine

KW - Expression

KW - Flavoenzyme

KW - Histidine tag

KW - Purification

KW - RNA methyltransferase

KW - Recombinant TrmFO protein

UR - https://www.scopus.com/pages/publications/77955415071

U2 - 10.1016/j.pep.2010.04.013

DO - 10.1016/j.pep.2010.04.013

M3 - Article

C2 - 20412857

AN - SCOPUS:77955415071

SN - 1046-5928

VL - 73

SP - 83

EP - 89

JO - Protein Expression and Purification

JF - Protein Expression and Purification

IS - 1