Cloning, expression, and purification of the His(6)-tagged thermostable beta-galactosidase from Pyrococcus woesei in Escherichia coli and some properties of the isolated enzyme.
In the previous study we cloned Pyrococcus woesei gene coding thermostable beta-galactosidase into pET30-LIC expression plasmid. The nucleotide sequence revealed that beta-galactosidase of P. woesei consists of 510 amino acids and has a molecular weight of 59, 056 kDa (GenBank Accession No. AF043283). It shows 99.9% nucleotide identity to the nucleotide sequence of beta-galactosidase from Pyrococcus furiosus. We also demonstrated that thermostable beta-galactosidase can be produced with high yield by Escherichia coli strain and can be easy separated by thermal precipitation of other bacterial proteins at 85 degrees C (S. D $$;abrowski, J. Maciuńska, and J. Synowiecki, 1998, Mol. Biotechnol. 10, 217-222). In this study we presented a new expression system for producing P. woesei beta-galactosidase in Escherichia coli and one-step chromatography purification procedure for obtaining pure enzyme (His(6)-tagged beta-galactosidase). The recombinant beta-galactosidase contained a polyhistidine tag at the N-terminus (20 additional amino acids) that allowed single-step isolation by Ni affinity chromatography. The enzyme was purified by heat treatment (to denature E. coli proteins), followed by metal-affinity chromatography on Ni(2+)-TED-Sepharose columns. The enzyme was characterized and displayed high activity and thermostability. This bacterial expression system appears to be a good method for production of the thermostable beta-galactosidase.
Daabrowski
S
S
Department of Microbiology, Technical University of Gdańsk, ul. Narutowicza 11/12, Gdańsk, 80-952, Poland.
Sobiewska
G
G
Maciuńska
J
J
Synowiecki
J
J
Kur
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D002847:Chromatography, Agarose; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006361:Heating; D006639:Histidine / Q000737:chemistry; D008969:Molecular Sequence Data; D009532:Nickel; D019711:Pyrococcus / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012685:Sepharose / Q000031:analogs & derivatives; D001616:beta-Galactosidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D006639:Histidine; D009532:Nickel; C039303:tris(carboxymethyl)ethylenediamine-sepharose; D012685:Sepharose; D001616:beta-Galactosidase