Molecular cloning, characterization, and expression of a cDNA coding copper/zinc superoxide dismutase from black porgy

Chi Tsai Lin; Tung Liang Lee; Kow Jen Duan; Chuian Fu Kent

doi:10.1021/jf000297k

Molecular cloning, characterization, and expression of a cDNA coding copper/zinc superoxide dismutase from black porgy

Chi Tsai Lin, Tung Liang Lee, Kow Jen Duan, Chuian Fu Kent

Department of Biology

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

14 Citations (Scopus)

Abstract

A full-length complementary DNA (cDNA) clone encoding a putative copper/zinc superoxide dismutase (Cu/Zn-SOD) was amplified by a Polymerase Chain Reaction (PCR) based technique from cDNA synthesized from black porgy, Acanthopagrus schlegeli, mRNA. Nucleotide sequence analysis of this cDNA clone revealed that it comprised a complete open reading frame coding for 154 amino acid residues. The deduced amino acid sequence showed slightly higher identity (72.8-78.1%) with shark and swordfish Cu/Zn-SOD than with Cu/Zn-SOD from mammalian (68.1-70.7%) and plant (55.5-56.5%) sources. The residues required for coordinating copper and zinc are conserved as they are among all reported Cu/Zn-SOD sequences. The deduced amino acid sequence lacks mitochondria targeting sequence, which suggests that the black porgy cDNA clone encodes a cytosolic Cu/Zn-SOD. The coding region of Cu/Zn-SOD from black porgy was introduced into an expression vector, pET-20b(+), and transformed into Escherichia coli AD494(DE3)pLysS. A predominant achromatic zone was detected by activity staining of native PAGE. This indicates that the Cu/Zn-SOD cDNA clone can express active Cu/Zn-SOD enzyme in E. coli.

Original language	English
Pages (from-to)	4444-4447
Number of pages	4
Journal	Journal of Agricultural and Food Chemistry
Volume	48
Issue number	9
DOIs	https://doi.org/10.1021/jf000297k
Publication status	Published - 2000 Oct 13

All Science Journal Classification (ASJC) codes

Chemistry(all)
Agricultural and Biological Sciences(all)

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10.1021/jf000297k

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@article{359bbd288dc5470daf8d5c5fdf29aae7,

title = "Molecular cloning, characterization, and expression of a cDNA coding copper/zinc superoxide dismutase from black porgy",

abstract = "A full-length complementary DNA (cDNA) clone encoding a putative copper/zinc superoxide dismutase (Cu/Zn-SOD) was amplified by a Polymerase Chain Reaction (PCR) based technique from cDNA synthesized from black porgy, Acanthopagrus schlegeli, mRNA. Nucleotide sequence analysis of this cDNA clone revealed that it comprised a complete open reading frame coding for 154 amino acid residues. The deduced amino acid sequence showed slightly higher identity (72.8-78.1%) with shark and swordfish Cu/Zn-SOD than with Cu/Zn-SOD from mammalian (68.1-70.7%) and plant (55.5-56.5%) sources. The residues required for coordinating copper and zinc are conserved as they are among all reported Cu/Zn-SOD sequences. The deduced amino acid sequence lacks mitochondria targeting sequence, which suggests that the black porgy cDNA clone encodes a cytosolic Cu/Zn-SOD. The coding region of Cu/Zn-SOD from black porgy was introduced into an expression vector, pET-20b(+), and transformed into Escherichia coli AD494(DE3)pLysS. A predominant achromatic zone was detected by activity staining of native PAGE. This indicates that the Cu/Zn-SOD cDNA clone can express active Cu/Zn-SOD enzyme in E. coli.",

author = "Lin, {Chi Tsai} and Lee, {Tung Liang} and Duan, {Kow Jen} and Kent, {Chuian Fu}",

year = "2000",

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T1 - Molecular cloning, characterization, and expression of a cDNA coding copper/zinc superoxide dismutase from black porgy

AU - Lin, Chi Tsai

AU - Lee, Tung Liang

AU - Duan, Kow Jen

AU - Kent, Chuian Fu

PY - 2000/10/13

Y1 - 2000/10/13

N2 - A full-length complementary DNA (cDNA) clone encoding a putative copper/zinc superoxide dismutase (Cu/Zn-SOD) was amplified by a Polymerase Chain Reaction (PCR) based technique from cDNA synthesized from black porgy, Acanthopagrus schlegeli, mRNA. Nucleotide sequence analysis of this cDNA clone revealed that it comprised a complete open reading frame coding for 154 amino acid residues. The deduced amino acid sequence showed slightly higher identity (72.8-78.1%) with shark and swordfish Cu/Zn-SOD than with Cu/Zn-SOD from mammalian (68.1-70.7%) and plant (55.5-56.5%) sources. The residues required for coordinating copper and zinc are conserved as they are among all reported Cu/Zn-SOD sequences. The deduced amino acid sequence lacks mitochondria targeting sequence, which suggests that the black porgy cDNA clone encodes a cytosolic Cu/Zn-SOD. The coding region of Cu/Zn-SOD from black porgy was introduced into an expression vector, pET-20b(+), and transformed into Escherichia coli AD494(DE3)pLysS. A predominant achromatic zone was detected by activity staining of native PAGE. This indicates that the Cu/Zn-SOD cDNA clone can express active Cu/Zn-SOD enzyme in E. coli.

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