Purification and characterization of yeast orotidine 5'-monophosphate decarboxylase overexpressed from plasmid PGU2

J.B Bell,M.E Jones

doi:10.1016/s0021-9258(18)98950-7

Abstract

Orotidine 5'-monophosphate decarboxylase (ODCase) has been overexpressed in yeast 15C cells transformed with a plasmid carrying the URA3 gene that encodes ODCase. Twenty g of cells having ODCase activity equal to 30 mg of pure enzyme per liter of cell culture were obtained after 9 h of galactose induction. To remove yeast proteases, a 60-90% ammonium sulfate fractionation step plus the addition of EDTA as an inhibitor of metallopeptidases was necessary. The purification protocol yielded ODCase that was protease-free and stable to storage at 4 degrees C for 16 months. The pure enzyme had a specific activity of 40 units/mg in 50 mM phosphate buffer, pH 6, and could be stored at -20 degrees C in 20% glycerol with retention of full activity for more than 2 years. The enzyme had a Km for orotidine 5'-monophosphate of 0.7 microM at pH 6 and 25 degrees C. The molecular weight of the plasmid-derived ODCase monomer determined by electrophoresis on denaturing polyacrylamide gels was 29,500. ODCase sedimented through sucrose density gradients as a monomer of about 30 kDa at low protein concentration and in the absence of ligands that bind at the catalytic site. An increase in the sedimentation rate could be induced by increasing the ODCase concentration or by adding ligands that are competitive inhibitors. ODCase sedimented in a single band typical of a protein of 46 kDa at the highest protein concentration studied or in the presence of 50 mM phosphate or 933 microM substrate (orotidine 5'-monophosphate) or product (UMP). A dimer sedimenting as a protein of about 64 kDa occurred in the presence of 50 microM 6-azauridine 5'-monophosphate or 2 microM 1-(5'-phospho-beta-D-ribofuranosyl) barbituric acid, competitive inhibitors of ODCase. These results resemble the ligand-induced subunit association of the ODCase domain of bifunctional UMP synthase and support the use of yeast ODCase as a model for ODCases from other species.

Highlights

In the1970’sODCase was purified from commercial bakers’ yeast by several groups[6,7,8]
TheGAL4 protein promotes transcription from the promoter for GALl in response to galactose, allowing the expression of plasmid-derived proteins to be induced by adding galactose to thegrowth medium
There- eliminated the remaining neutral baansidc protease activities, fore, modifications to their published procedure for overexpression of yeast ODCase fromstrain Sf657-2D cells carrying the pGU2 plasmid [9] were necessary to obtain a high level of ODCase expression in yeast strai1n5C

Summary

Introduction

In the1970’sODCase was purified from commercial bakers’ yeast by several groups[6,7,8]. We have added 2 mM EDTA to the protease inhibitor mixture containing 1 mM PMSF, 1 PM pepstatin A, and 0.6 pM leupeptin andhave routinely addedthis mixture to eabcuhffer the total ODCase activity occurred when cells grown in unsupplemented YPmedium were harvested after 2 h of galactose induction (Table I).

Results

Conclusion