1. Isolation and expression in Escherichia coli of a cDNA clone encoding human β-glucuronidase
- Author
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R.A. Gravel, Palmer R, F. Quan, Lamhonwah Am, John S. Waye, K S Guise, Ganschow Re, William S. Sly, and R G Korneluk
- Subjects
DNA, Recombinant ,Molecular cloning ,medicine.disease_cause ,Homology (biology) ,Restriction fragment ,Mice ,Restriction map ,Species Specificity ,Complementary DNA ,Escherichia coli ,Genetics ,medicine ,Animals ,Humans ,Amino Acid Sequence ,Cloning, Molecular ,Glucuronidase ,Base Sequence ,biology ,cDNA library ,General Medicine ,Mucopolysaccharidoses ,Molecular biology ,Open reading frame ,Gene Expression Regulation ,biology.protein ,Plasmids - Abstract
Mucopolysaccharidosis type VII is a lysosomal storage disease resulting from a deficiency of β-glucuronidase (BG) activity. To facilitate the investigation of mutation in the disease and provide molecular diagnostic tools for affected families, we have isolated human BG cDNA clones. The SV40-transformed human fibroblast cDNA library of Okayama and Berg [Mol. Cell. Biol. 3 (1982) 280–289] was screened with a fragment of a murine BG cDNA clone (pGUS-1). The 17 human cDNA clones (pHUG) isolated were identical by restriction mapping, varying only in length. The pHUG clones show 80% DNA sequence homology with pGUS-1 in a 198-bp PvuII-SstI restriction fragment. Both pGUS-1 and the pHUG clones contained an open reading frame (ORF) throughout the sequenced region with a predicted amino acid sequence homology of 73 %. Expression in Escherichia coli of a 1150-bp fragment of pHUG-1 subcloned in pUC9 resulted in an isopropylthio -β-galactoside (IPTG)-inducible 35-kDal fusion protein which was specifically immunoprecipitated by goat anti-human BG immunoglobulin G (IgG). This evidence provides direct confirmation that the pHUG cDNA clones correspond to human BG.
- Published
- 1985