Chemically-synthesized gene encoding modified human superoxide dismutase: its construction, expression and properties of the product.
The gene encoding modified human superoxide dismutase (h-SOD) with 153 amino acid residues was constructed by chemical synthesis using the phosphoramidite method. The gene was designed so as to use bacterial codons for expression in prokaryotes and to introduce several unique restriction sites for further mutagenesis by the cassette exchange method. The distance between Shine-Dalgarno sequence and initiation codon was adjusted to maximum expression by using synthesized oligonucleotide. In addition, Cys 6 of h-SOD was changed to Ala to improve instability of native h-SOD. Synthesized structural gene of h-SOD was expressed in E. coli after induction of isopropyl beta-D-thiogalactoside by inserting the gene into the expression vector pKK223-3 having tac promoter. The gene that has 10 base pairs between Shine-Dalgarno sequence and initiation codon showed the most efficient expression. The gene produced three active SOD isomers as revealed by chromatofocusing. The main isomer was purified to homogeneity and characterized. The h-SOD-Ala6 showed similar properties to those of native h-SOD with respect to molecular weight, subunit structure, absorption spectrum, but the modified SOD was more resistant to heat denaturation than was native h-SOD; half-denaturing temperature was shifted by 10 degrees C. Thus, the exchange of Cys 6 to Ala of h-SOD increased a stability of the enzyme.
Kanematsu
S
S
Hagiwara Institute of Health, Hyogo, Japan.
Takeshima
Y
Y
Hagiwara
H
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D004795:Enzyme Stability; D015870:Gene Expression; D005796:Genes; D005813:Genes, Synthetic; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D013482:Superoxide Dismutase / Q000737:chemistry / Q000235:genetics*
D013482:Superoxide Dismutase