Expression of the human alpha-galactosidase A in Escherichia coli K-12.
We used the prokaryotic expression vector, ptrpL1, for the expression in Escherichia coli K-12 of a cDNA clone specific for the human lysosomal hydrolase, alpha-galactosidase A. The 5' terminus of the cDNA clone was engineered so that an ATG codon precedes the first codon of the mature form of the enzyme. A clone with elevated expression of this human enzyme was constructed by increasing the distance between the Shine-Dalgarno site and the ATG start codon from 6 to 8 bp. Clones with alpha-galactosidase A specific cDNA encoding the proenzyme produce a protein of 45 kDa, the size expected for the intact proenzyme. The 45-kDa protein is specifically precipitated by antibody to alpha-galactosidase A, and its expression is repressed by tryptophan and induced by 3-beta-indoleacrylic acid as expected for this expression vector. The human enzyme is produced in E. coli in a catalytically active form at levels sufficient to support the growth of cells using alpha-galactosides as sole sources of carbon and energy. In addition, bacterial colonies that produce the human enzyme turn blue in the presence of 5-bromo-4-chloro-3-indolyl-alpha-D-galactopyranoside.
Hantzopoulos
P A
PA
Department of Microbiology, Mount Sinai School of Medicine, New York, NY 10029.
Calhoun
D H
DH
D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D000795:Fabry Disease / Q000188:drug therapy; D005696:Galactosidases / Q000235:genetics*; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006801:Humans; D010957:Plasmids; D000519:alpha-Galactosidase / Q000096:biosynthesis / Q000235:genetics*
D004247:DNA; D005696:Galactosidases; D000519:alpha-Galactosidase