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

Research output: Contribution to journal › Article › peer-review

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.

Original language	English
Pages (from-to)	83-89
Number of pages	7
Journal	Protein Expression and Purification
Volume	73
Issue number	1
DOIs	https://doi.org/10.1016/j.pep.2010.04.013
Publication status	Published - 1 Jan 2010

Keywords

5-Methyluridine
Expression
Flavoenzyme
Histidine tag
Purification
RNA methyltransferase
Recombinant TrmFO protein

Access to Document

10.1016/j.pep.2010.04.013

Cite this

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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.",

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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

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

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