Gene cloning, expression and purification of human mitochondrial tRNALeu(UUR) and its mutant

Han Weiguo; Chen Li; Liu Jing; Zha Xiliang; Jin Youxin; Wang Debao

Gene cloning, expression and purification of human mitochondrial tRNA^Leu(UUR) and its mutant

Han Weiguo, Chen Li, Liu Jing, Zha Xiliang, Jin Youxin, Wang Debao

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

Abstract

Genes of human mitochondrial tRNA^Leu(UUR) (mtRNA^Leu(UUR)) and its mutant (mtRNA^Leu(M)) were synthesized and inserted into the plasmid pGEM-9Zf(-) respectively. E.coli JM 109 was transformed by the recombinant plasmids containing the target genes. The mtRNA^Leu(UUR) and mtRNA^Leu(M) were expressed up to 19.10% and 17.76% of total small RNA respectively. They were purified to 54% homogeneity by DEAE-sepharose-CL4B column chromatography and finally repurified by 15% PAGE/urea. Their kinetic parameters for E.coli LeuRS were measured. The results showed that the value of k_cal/ K_m of mtRNA^Leu(M) was about one fifth of that of mtRNA^Leu(UUR) and indicated the leucine acceptability of mtRNA^Leu(M) was much lower than that of mtRNA^Leu(UUR).

Original language	English (US)
Pages (from-to)	X-120
Journal	Science in China, Series C: Life Sciences
Volume	44
Issue number	2
State	Published - Apr 2001
Externally published	Yes

Keywords

Expression
Gene cloning
Human mitochondrial tRNA
Mutation
Purification

ASJC Scopus subject areas

Renewable Energy, Sustainability and the Environment
Modeling and Simulation
General Biochemistry, Genetics and Molecular Biology
Fuel Technology
General Environmental Science
Energy Engineering and Power Technology
General Agricultural and Biological Sciences
General Energy
Management, Monitoring, Policy and Law

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

Cite this

@article{72e4d60a022d431ba9eb30da64de873d,

title = "Gene cloning, expression and purification of human mitochondrial tRNALeu(UUR) and its mutant",

abstract = "Genes of human mitochondrial tRNALeu(UUR) (mtRNALeu(UUR)) and its mutant (mtRNALeu(M)) were synthesized and inserted into the plasmid pGEM-9Zf(-) respectively. E.coli JM 109 was transformed by the recombinant plasmids containing the target genes. The mtRNALeu(UUR) and mtRNALeu(M) were expressed up to 19.10% and 17.76% of total small RNA respectively. They were purified to 54% homogeneity by DEAE-sepharose-CL4B column chromatography and finally repurified by 15% PAGE/urea. Their kinetic parameters for E.coli LeuRS were measured. The results showed that the value of kcal/ Km of mtRNALeu(M) was about one fifth of that of mtRNALeu(UUR) and indicated the leucine acceptability of mtRNALeu(M) was much lower than that of mtRNALeu(UUR).",

keywords = "Expression, Gene cloning, Human mitochondrial tRNA, Mutation, Purification",

author = "Han Weiguo and Chen Li and Liu Jing and Zha Xiliang and Jin Youxin and Wang Debao",

year = "2001",

month = apr,

language = "English (US)",

volume = "44",

pages = "X--120",

journal = "Science in China, Series C: Life Sciences",

issn = "1006-9305",

publisher = "Science in China Press",

number = "2",

}

TY - JOUR

T1 - Gene cloning, expression and purification of human mitochondrial tRNALeu(UUR) and its mutant

AU - Weiguo, Han

AU - Li, Chen

AU - Jing, Liu

AU - Xiliang, Zha

AU - Youxin, Jin

AU - Debao, Wang

PY - 2001/4

Y1 - 2001/4

N2 - Genes of human mitochondrial tRNALeu(UUR) (mtRNALeu(UUR)) and its mutant (mtRNALeu(M)) were synthesized and inserted into the plasmid pGEM-9Zf(-) respectively. E.coli JM 109 was transformed by the recombinant plasmids containing the target genes. The mtRNALeu(UUR) and mtRNALeu(M) were expressed up to 19.10% and 17.76% of total small RNA respectively. They were purified to 54% homogeneity by DEAE-sepharose-CL4B column chromatography and finally repurified by 15% PAGE/urea. Their kinetic parameters for E.coli LeuRS were measured. The results showed that the value of kcal/ Km of mtRNALeu(M) was about one fifth of that of mtRNALeu(UUR) and indicated the leucine acceptability of mtRNALeu(M) was much lower than that of mtRNALeu(UUR).

AB - Genes of human mitochondrial tRNALeu(UUR) (mtRNALeu(UUR)) and its mutant (mtRNALeu(M)) were synthesized and inserted into the plasmid pGEM-9Zf(-) respectively. E.coli JM 109 was transformed by the recombinant plasmids containing the target genes. The mtRNALeu(UUR) and mtRNALeu(M) were expressed up to 19.10% and 17.76% of total small RNA respectively. They were purified to 54% homogeneity by DEAE-sepharose-CL4B column chromatography and finally repurified by 15% PAGE/urea. Their kinetic parameters for E.coli LeuRS were measured. The results showed that the value of kcal/ Km of mtRNALeu(M) was about one fifth of that of mtRNALeu(UUR) and indicated the leucine acceptability of mtRNALeu(M) was much lower than that of mtRNALeu(UUR).

KW - Expression

KW - Gene cloning

KW - Human mitochondrial tRNA

KW - Mutation

KW - Purification

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M3 - Article

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SN - 1006-9305

VL - 44

SP - X-120

JO - Science in China, Series C: Life Sciences

JF - Science in China, Series C: Life Sciences

IS - 2

ER -