Cloning and expression of the beta-galactosidase genes from Lactobacillus reuteri in Escherichia coli.
Heterodimeric beta-galactosidase of Lactobacillus reuteri L103 is encoded by two overlapping genes, lacL and lacM. The lacL (1887bp) and lacM (960bp) genes encode polypeptides with calculated molecular masses of 73,620 and 35,682Da, respectively. The deduced amino acid sequences of lacL and lacM show significant identity with the sequences of beta-galactosidases from other lactobacilli and Escherichia coli. The coding regions of the lacLM genes were cloned and successfully overexpressed in E. coli using an expression system based on the T7 RNA polymerase promoter. Expression of lacL alone and coexpression of lacL and lacM as well as activity staining of both native and recombinant beta-galactosidases suggested a translational coupling between lacL and lacM, indicating that the formation of a functional beta-galactosidase requires both genes. Recombinant beta-galactosidase was purified to apparent homogeneity, characterized and compared with the native beta-galactosidase from L. reuteri L103.
Nguyen
Thu-Ha
TH
Research Centre Applied Biocatalysis, Petersgasse 14, Graz, Austria.
Splechtna
Barbara
B
Yamabhai
Montarop
M
Haltrich
Dietmar
D
Peterbauer
Clemens
C
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics*; D005784:Gene Amplification; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D052200:Limosilactobacillus reuteri / Q000201:enzymology* / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000302:isolation & purification; D001616:beta-Galactosidase / Q000235:genetics* / Q000302:isolation & purification
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D001616:beta-Galactosidase