Isolation of cDNA clones for human beta-glucocerebrosidase using the lambda gt11 expression system

Abstract

Two cDNA clones (lambda GC-1 and lambda GC-2) for human beta-glucocerebrosidase [EC 3.2.1.45] have been isolated from a human hepatoma library in lambda gt11 by immunological screening using monospecific polyclonal antibody for beta-glucocerebrosidase. Restriction endonuclease mapping indicates that these clones are probably identical in size, each with a 1900 bp insert. The 50 kDa size of the insert-encoded polypeptide produced by these clones in fusion with beta-galactosidase of lambda gt11 in E. coli BNN103 is consistent with the size of the nascent form of beta-glucocerebrosidase. These fusion proteins are shown by Western blotting to react with antibody to beta-glucocerebrosidase. Amino acid sequence deduced from the nucleotide sequence of the insert ir pGC-1 is identical to known amino acid sequence of beta-glucocerebrosidase, and thus, confirms that the clones are specific for beta-glucocerebrosidase.