D-Xylulose kinase from Saccharomyces cerevisiae: isolation and characterization of the highly unstable enzyme, recombinantly produced in Escherichia coli.

ABSTRACT: The Saccharomyces cerevisiae gene encoding xylulose kinase (XKS1) was over-expressed to an abundance of ≥ 10% intracellular protein in Escherichia coli. Instability of XKS1, not pointed out in previous reports of the enzyme, prevented isolation of active enzyme in native or "tagged" form under a wide range of purification conditions. A fusion protein haboring C-terminal Strep-tag II (XKS1-Strep) displayed activity (∼20 U/mg) as isolated. However, the half-life time of purified XKS1-Strep was only ∼1.5h at 4°C and could not be enhanced substantially by an assortment of extrinsic stabilizers (osmolytes, protein, substrates). Peptide mass mapping and N-terminal sequencing showed that the recombinant protein was structurally intact, ruling out proteolytic processing and chemical modifications

SUBMITTER: Pival SL 

PROVIDER: S-EPMC3158326 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature