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 sequen ce revealed that beta-galactosidase of P. woesei consists of 510 amino acid s 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-gal actosidase from Pyrococcus furiosus. We also demonstrated that thermostable beta-galactosidase can be produced with high yield by Escherichia coli str ain and can be easy separated by thermal precipitation of other bacterial p roteins at 85 degrees C (S. Dabrowski, J, Maciunska, and J, Synowiecki, 199 8, Mol. Biotechnol; 10, 217-222), In this study we presented a new expressi on system for producing P. woesei beta-galactosidase in Escherichia coli an d one-step chromatography purification procedure for obtaining pure enzyme (His(6)-tagged beta-galactosidase), The recombinant beta-galactosidase cont ained a polyhistidine tag at the N-terminus (20 additional amino acids) tha t allowed single-step isolation by Ni affinity chromatography, The enzyme w as purified by heat treatment (to denature E. coli proteins), followed by m etal-affinity chromatography on Ni2+-TED-Sepharose columns. The enzyme was characterized and displayed high activity and thermostability, This bacteri al expression system appears to be a good method for production of the ther mostable beta-galactosidase. (C) 2000 Academic Press.