High-level expression and single-step purification of leucyl-tRNA synthetase from Aquifex aeolicus

Chen Ling; Yong Gang Zheng; En Duo Wang

doi:10.1016/j.pep.2004.04.003

High-level expression and single-step purification of leucyl-tRNA synthetase from Aquifex aeolicus

Chen Ling, Yong Gang Zheng, En Duo Wang

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

1 Scopus citations

Abstract

Aquifex aeolicus leucyl-tRNA synthetase is the only known heterodimeric LeuRS, consisting of two subunits with molecular masses of 74.0 and 33.5kDa, and named αβ-LeuRS. The gene encoding α subunit was cloned into pSBET-b vector. Synthetic oligonucleotide encoding six histidine residues was also inserted in front of α subunit. PSBET-b vector contains argU gene, which encodes a rare Escherichia coli tRNA^Arg(AGA/AGG). The argU gene helps A. aeolicus LeuRS, which contains AGA/AGG codons in exceptionally high frequency, express well in E. coli. The gene encoding β subunit was inserted into pET-15b vector. E. coli BL21-CodonPlus (DE3) cells were transformed with the two recombinant plasmids to produce αβ-LeuRS with a His₆ tag at the N-terminus of α subunit. The enzyme was purified by affinity chromatography on Ni-NTA Superflow. About 7mg purified αβ-LeuRS was obtained from 250ml culture. The His₆-tag at the N-terminus did not affect the aminoacylation activity of the enzyme.

Original language	English (US)
Pages (from-to)	146-149
Number of pages	4
Journal	Protein Expression and Purification
Volume	36
Issue number	1
DOIs	https://doi.org/10.1016/j.pep.2004.04.003
State	Published - Jul 2004
Externally published	Yes

Keywords

Aquifex aeolicus
Escherichia coli
Leucyl-tRNA synthetase
Overproduction
argU

ASJC Scopus subject areas

Biotechnology

Access to Document

10.1016/j.pep.2004.04.003

Cite this

@article{251e817e720e4e8faaa9dd1488adb734,

title = "High-level expression and single-step purification of leucyl-tRNA synthetase from Aquifex aeolicus",

abstract = "Aquifex aeolicus leucyl-tRNA synthetase is the only known heterodimeric LeuRS, consisting of two subunits with molecular masses of 74.0 and 33.5kDa, and named αβ-LeuRS. The gene encoding α subunit was cloned into pSBET-b vector. Synthetic oligonucleotide encoding six histidine residues was also inserted in front of α subunit. PSBET-b vector contains argU gene, which encodes a rare Escherichia coli tRNAArg(AGA/AGG). The argU gene helps A. aeolicus LeuRS, which contains AGA/AGG codons in exceptionally high frequency, express well in E. coli. The gene encoding β subunit was inserted into pET-15b vector. E. coli BL21-CodonPlus (DE3) cells were transformed with the two recombinant plasmids to produce αβ-LeuRS with a His6 tag at the N-terminus of α subunit. The enzyme was purified by affinity chromatography on Ni-NTA Superflow. About 7mg purified αβ-LeuRS was obtained from 250ml culture. The His6-tag at the N-terminus did not affect the aminoacylation activity of the enzyme.",

keywords = "Aquifex aeolicus, Escherichia coli, Leucyl-tRNA synthetase, Overproduction, argU",

author = "Chen Ling and Zheng, {Yong Gang} and Wang, {En Duo}",

note = "Funding Information: We are grateful to Professor You-Shang Zhang for carefully reading the manuscript. This work was funded by Chinese Natural Sciences Foundation (Grants 30170224 and 30330180), 863 projects of China (Grant 2001AA235071), and Shanghai Committee of Science and Technology (Grant No. 02DJ140567). Copyright: Copyright 2008 Elsevier B.V., All rights reserved.",

year = "2004",

month = jul,

doi = "10.1016/j.pep.2004.04.003",

language = "English (US)",

volume = "36",

pages = "146--149",

journal = "Protein Expression and Purification",

issn = "1046-5928",

publisher = "Academic Press Inc.",

number = "1",

}

TY - JOUR

T1 - High-level expression and single-step purification of leucyl-tRNA synthetase from Aquifex aeolicus

AU - Ling, Chen

AU - Zheng, Yong Gang

AU - Wang, En Duo

N1 - Funding Information: We are grateful to Professor You-Shang Zhang for carefully reading the manuscript. This work was funded by Chinese Natural Sciences Foundation (Grants 30170224 and 30330180), 863 projects of China (Grant 2001AA235071), and Shanghai Committee of Science and Technology (Grant No. 02DJ140567). Copyright: Copyright 2008 Elsevier B.V., All rights reserved.

PY - 2004/7

Y1 - 2004/7

N2 - Aquifex aeolicus leucyl-tRNA synthetase is the only known heterodimeric LeuRS, consisting of two subunits with molecular masses of 74.0 and 33.5kDa, and named αβ-LeuRS. The gene encoding α subunit was cloned into pSBET-b vector. Synthetic oligonucleotide encoding six histidine residues was also inserted in front of α subunit. PSBET-b vector contains argU gene, which encodes a rare Escherichia coli tRNAArg(AGA/AGG). The argU gene helps A. aeolicus LeuRS, which contains AGA/AGG codons in exceptionally high frequency, express well in E. coli. The gene encoding β subunit was inserted into pET-15b vector. E. coli BL21-CodonPlus (DE3) cells were transformed with the two recombinant plasmids to produce αβ-LeuRS with a His6 tag at the N-terminus of α subunit. The enzyme was purified by affinity chromatography on Ni-NTA Superflow. About 7mg purified αβ-LeuRS was obtained from 250ml culture. The His6-tag at the N-terminus did not affect the aminoacylation activity of the enzyme.

AB - Aquifex aeolicus leucyl-tRNA synthetase is the only known heterodimeric LeuRS, consisting of two subunits with molecular masses of 74.0 and 33.5kDa, and named αβ-LeuRS. The gene encoding α subunit was cloned into pSBET-b vector. Synthetic oligonucleotide encoding six histidine residues was also inserted in front of α subunit. PSBET-b vector contains argU gene, which encodes a rare Escherichia coli tRNAArg(AGA/AGG). The argU gene helps A. aeolicus LeuRS, which contains AGA/AGG codons in exceptionally high frequency, express well in E. coli. The gene encoding β subunit was inserted into pET-15b vector. E. coli BL21-CodonPlus (DE3) cells were transformed with the two recombinant plasmids to produce αβ-LeuRS with a His6 tag at the N-terminus of α subunit. The enzyme was purified by affinity chromatography on Ni-NTA Superflow. About 7mg purified αβ-LeuRS was obtained from 250ml culture. The His6-tag at the N-terminus did not affect the aminoacylation activity of the enzyme.

KW - Aquifex aeolicus

KW - Escherichia coli

KW - Leucyl-tRNA synthetase

KW - Overproduction

KW - argU

UR - http://www.scopus.com/inward/record.url?scp=3042598416&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=3042598416&partnerID=8YFLogxK

U2 - 10.1016/j.pep.2004.04.003

DO - 10.1016/j.pep.2004.04.003

M3 - Article

C2 - 15177296

AN - SCOPUS:3042598416

SN - 1046-5928

VL - 36

SP - 146

EP - 149

JO - Protein Expression and Purification

JF - Protein Expression and Purification

IS - 1

ER -

High-level expression and single-step purification of leucyl-tRNA synthetase from Aquifex aeolicus

Abstract

Keywords

ASJC Scopus subject areas

Access to Document

Other files and links

Fingerprint

Cite this