tailieunhanh - Báo cáo Y học: Overexpression of a recombinant wild-type and His-tagged Bacillus subtilis glycine oxidase in Escherichia coli

We have cloned the gene coding for theBacillus subtilis glycine oxidase (GO), a new ¯avoprotein that oxidizes gly-cine and sarcosine to the corresponding a-keto acid, ammonia and hydrogen peroxide. By inserting the DNA encoding for GO into the multiple cloning site of the (pT7-GO).ThepT7-GOencodes a fullyactive fusionprotein with six additional residues at the N-terminus of GO (MARIRA). | Eur. J. Biochem. 269 1456-1463 2002 FEBS 2002 Overexpression of a recombinant wild-type and His-tagged Bacillus subtilis glycine oxidase in Escherichia coli Viviana Job Gianluca Molla Mirella S. Pilone and Loredano Pollegioni Department of Structural and Functional Biology University of Insubria Varese Italy We have cloned the gene coding for the Bacillus subtilis glycine oxidase GO a new flavoprotein that oxidizes glycine and sarcosine to the corresponding a-keto acid ammonia and hydrogen peroxide. By inserting the DNA encoding for GO into the multiple cloning site of the expression vector we produced a recombinant plasmid pT7-GO . The pT7-GO encodes a fully active fusion protein with six additional residues at the N-terminus of GO MARIRA . In BL21 DE3 pLysS Escherichia coli cells and under optimal isopropyl thio-b-D-galactoside induction conditions soluble and active chimeric GO was expressed up to U g-1 of cell and a fermentation yield of U-L-1 of fermentation broth . An N-terminal His-tagged protein HisGO was also successfully expressed in E. coli as a soluble protein and a fully active holoenzyme. HisGO represents w of the total soluble protein content of the cell. The His-tagged GO was purified in a single step by nickel-chelate chromatography to a specific activity of U-mg-1 protein at 25 C and with a yield of 98 . The characterization of the purified enzyme showed that GO is a homotetramer of w 180 kDa with the spectral properties typical of flavoproteins. GO exhibits good thermal stability with a Tm of 46 C after 30 min incubation its stability is maximal in the pH range. A comparison of aminoacid sequence and substrate specificity indicates that GO has similarities to other flavoenzymes acting on primary amines and on D-amino acids. Keywords glycine oxidase flavoprotein oxidase His-tagged protein purification. Glycine oxidase GO is a flavoenzyme that was recently discovered following the complete sequencing of the Bacillus