High gene expression in Escherichia coli of recombinant alginate lyase as a fused protein with beta-galactosidase alpha-peptide.
Escherichia coli LE392 (pAL28) was previously isolated as a positive clone harboring the alginate lyase gene (aly) from an alginate-degrading strain, Pseudomonas sp. OS-ALG-9. The plasmid pAL205, one of the constructs obtained after successive subcloning of pAL28, gave the highest expression of aly in E. coli cells. A 8-fold increase in the alginate lyase (Aly) activity in E. coli JM109 (pAL205) was induced with isopropyl-beta-D-thiogalactoside, which was 210 times higher than that in E. coli LE392 (pAL28). The highly significant increase in the expression of the Aly enzyme with pAL205 was investigated through the nucleotide sequence around the 5' region of aly as well as the N-terminal sequence of the purified enzyme. It was found that the Aly expressed in E. coli (pAL205) was a fused protein containing 7 residues from the N-terminus of beta-galactosidase alpha-peptide and the mature protein found in the Pseudomonas sp. except for three residues in the N-terminal.
Fujiyama
K
K
International Center of Cooperative Research in Biotechnology, Faculty of Engineering, Osaka University, Japan.
Maki
H
H
Kinoshita
S
S
Yoshida
T
T
D000464:Alginates / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D011133:Polysaccharide-Lyases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D001616:beta-Galactosidase
D000464:Alginates; D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase; D011133:Polysaccharide-Lyases; C016158:poly(beta-D-mannuronate) lyase