Expression of human extracellular superoxide dismutase in Chinese hamster ovary cells and characterization of the product.
A complementary DNA clone from human placenta, encoding human extracellular superoxide dismutase (EC-SOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1), has recently been isolated and characterized. An expression plasmid, based on the EC-SOD complementary DNA, was transfected into Chinese hamster ovary cells (CHO-K1). The transfected cells secreted human EC-SOD to the culture medium. The secreted recombinant (r) EC-SOD was isolated in high yield with a three-step procedure beginning with immobilized monoclonal anti-EC-SOD antibodies. The properties of the rEC-SOD were compared with native (n) EC-SOD isolated from human umbilical cords. The specific activities and amino-terminal amino acid sequences were identical. The amino acid compositions were virtually identical and very similar to the composition deduced from the complementary DNA sequence. Both rEC-SOD and nEC-SOD contained 4 Cu and 4 Zn atoms per molecule, and the presence of Zn in EC-SOD is thus now established. The rEC-SOD produced is type C, since its affinity for heparin-Sepharose was identical to that of nEC-SOD type C. Both enzymes bound to concanavalin A, lentil lectin, and wheat germ lectin and are thus glycoproteins. rEC-SOD and nEC-SOD seem to have the same subunit structure and composition as analyzed by polyacrylamide gel electrophoresis and gel chromatography.
Tibell
L
L
Department of Molecular Genetics and Cell Biology, SYN-TEK AB, Umeå, Sweden.
Hjalmarsson
K
K
Edlund
T
T
Skogman
G
G
Engström
A
A
Marklund
S L
SL
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D004247:DNA / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D014176:Protein Biosynthesis; D013539:Simian virus 40 / Q000235:genetics; D013482:Superoxide Dismutase / Q000235:genetics*; D014158:Transcription, Genetic
D000596:Amino Acids; D004247:DNA; D013482:Superoxide Dismutase