Molecular Cloning and High-Level Expression of Human Cytoplasmic Superoxide Dismutase Gene in Escherichia coli

사람의 세포질 Superoxide Dismutase 유전자의 클로닝과 대장균내에서의 대량발현에 관한 연구

  • 이우길 (서울대학교 동물학과 분자유전학실험실) ;
  • 김영호 (서울대학교 동물학과 분자유전학실험실) ;
  • 양중익 (동아제약 연구소) ;
  • 노현모 (서울대학교 동물학과 분자유전학실험실)
  • Published : 1990.06.01

Abstract

Complementary DNA (cDNA) coding for human cytoplasmic superoxide dismutase (SOD1) (superoxide: superoxide oxidoreductase E.C.1.15.1.1) was isolated from human liver cDNA library of $\lambda$gt11 by in situ plaque hybridization. The insery cDNA gas the 5' untranslational region (UTR) and 3'UTR of SOD1 gene. Polymerase Chain Reaction (PCR) method was used fro subcloning of SOD1 structural gene. Using synthetic sense strand primer (24mer) containing a start codon and antisense strand primer (24mer), SOD1 structural gene was selectively amplified. Amplified DNA was directly cloned into the HincII site of pUC19 plasmid. Insery cDNA was subcloned into M13 mp19 and sequenced by dideowy chain termination method with Sequenase. The nucleotide sequence of insert cDNA had an open reading frame (ORF) coding for 153 amino acid residues. The structural gene of cytoplasmic SOD was placed under the control of bacteriophage $\lambda P_{L}$ regulatory sequences, generating a highly efficient expression plasmid. The production of human SOD1 in E. coli cells was about 7% of total cellular proteins and recombinant human SOD1 possessed its own enzymatic acitivity.

Keywords

Human SODI;Cloning Expression