Cloning, functional expression and purification of endo-beta-galactosidase from Flavobacterium keratolyticus.
Endo-beta-galactosidase (EC 3.2.1.103) is an enzyme that hydrolyzes internal endo-beta-galactosyl linkages in keratan sulfate, and glycoconjugates with N-acetyl-lactosamine repeating units. Here, we report the cloning of the endo-beta-galactosidase-encoding gene from Flavobacterium keratolyticus, its expression in Escherichia coli and the purification of the enzyme. The enzyme was purified over 15000-fold to apparent homogeneity. The purified endo-beta-galactosidase consists of a single band of about 43kDa on SDS-PAGE and has a specific activity of 148micro/mg. Based on peptide sequences derived from the purified enzyme, a full-length clone encoding endo-beta-galactosidase was isolated from F. keratolyticus genomic DNA. The gene contains a single open reading frame coding for a protein of 422 amino acid residues with a putative N-terminal signal peptide. Its authenticity was confirmed by colinearity of deduced amino acid sequences with the peptide sequences, and synthesis of enzyme in E. coli.
Leng
L
L
Cell Biochemistry Laboratory, Lindsley F. Kimball Research Institute, New York Blood Center, 310 East 67 Street, New York, NY 10021, USA.
Zhu
A
A
Zhang
Z
Z
Hurst
R
R
Goldstein
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005417:Flavobacterium / Q000201:enzymology* / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D006026:Glycoside Hydrolases / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D001616:beta-Galactosidase / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism
D017931:DNA Primers; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C026093:keratan-sulfate endo-1,4-beta-galactosidase; D001616:beta-Galactosidase