Using secretion to solve a solubility problem: high-yield expression in Escherichia coli and purification of the bacterial glycoamidase PNGase F

Protein Expr Purif. 2002 Feb;24(1):90-8. doi: 10.1006/prep.2001.1555.

Abstract

PNGase F is a widely used deglycosidase, secreted in small amounts by the gram-negative bacterium Flavobacterium meningosepticum. We have designed a T7 promoter-based Escherichia coli expression system to provide a high-yield source of recombinant enzyme. When expressed intracellularly, the enzyme was produced in a largely insoluble state. However, when expressed as a fusion with the leader sequence from the ompA gene, hexahistidine-tagged PNGase F was efficiently processed and exported to the E. coli periplasm. Single-step purification using immobilized metal affinity chromatography yielded 8 mg of pure enzyme per liter of culture, which is fully active on a range of protein and peptide substrates.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amidohydrolases / genetics*
  • Amidohydrolases / isolation & purification
  • Amidohydrolases / metabolism
  • Amino Acid Sequence
  • Bacterial Outer Membrane Proteins / genetics
  • Base Sequence
  • Cloning, Molecular / methods*
  • DNA, Complementary
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Flavobacterium / enzymology*
  • Flavobacterium / genetics
  • Gene Expression
  • Glycosylation
  • Mass Spectrometry
  • Molecular Sequence Data
  • Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / isolation & purification
  • Recombinant Fusion Proteins / metabolism
  • Solubility

Substances

  • Bacterial Outer Membrane Proteins
  • DNA, Complementary
  • Recombinant Fusion Proteins
  • OMPA outer membrane proteins
  • Amidohydrolases
  • Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase

Associated data

  • GENBANK/AF165910