Overexpression and purification of untagged polynucleotide phosphorylases

Protein Expr Purif. 2003 Dec;32(2):202-9. doi: 10.1016/j.pep.2003.08.005.

Abstract

We report here the development of new, straightforward procedures for the purification of bacterial polynucleotide phosphorylases (PNPases). The pnp genes from Streptomyces antibioticus, Streptomyces coelicolor, and Escherichia coli were overexpressed using the vectors pET11 and pET11A in E. coli BL21(DE3)pLysS. The enzymes were purified to apparent homogeneity after phosphorolysis in crude extracts followed by anion exchange and hydrophobic interaction chromatography. Yields of 5-15mg per liter of culture were obtained and the enzymes contained only small amounts of contaminating RNA as estimated from the A(280/260) ratios of purified preparations. All three enzymes were active in both the polymerization and phosphorolysis reactions normally catalyzed by PNPases. Incubation under phosphorolysis conditions but in the absence of potassium phosphate indicated that the enzymes were free of phosphate-independent nuclease activity. We suggest that the approaches described here may be applied generally to the overexpression and purification of eubacterial polynucleotide phosphorylases.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Adenosine Diphosphate / metabolism
  • Bacterial Proteins / genetics
  • Bacterial Proteins / isolation & purification
  • Bacterial Proteins / metabolism
  • Chromatography, Liquid / methods
  • Cloning, Molecular
  • DNA Primers / genetics
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Phosphates / chemistry
  • Polyribonucleotide Nucleotidyltransferase / biosynthesis*
  • Polyribonucleotide Nucleotidyltransferase / genetics
  • Polyribonucleotide Nucleotidyltransferase / isolation & purification*
  • Polyribonucleotide Nucleotidyltransferase / metabolism
  • Potassium Compounds / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Streptomyces / enzymology
  • Streptomyces / genetics
  • Streptomyces antibioticus / enzymology
  • Streptomyces antibioticus / genetics

Substances

  • Bacterial Proteins
  • DNA Primers
  • Phosphates
  • Potassium Compounds
  • Recombinant Proteins
  • Adenosine Diphosphate
  • potassium phosphate
  • Polyribonucleotide Nucleotidyltransferase