Expression, purification and characterisation of secreted recombinant glycoprotein PsA in Dictyostelium discoideum

J Biotechnol. 1995 Jan 15;38(2):137-49. doi: 10.1016/0168-1656(94)00127-x.

Abstract

Dictyostelium discoideum is a newly developed eukaryotic expression system which is an alternative to tissue cultures for the production of recombinant proteins requiring eukaryotic folding and post-translational modifications. The homologous glycoprotein PsA (prespore specific antigen) is a glycosyl phosphatidylinositol (GPI) anchored membrane protein from D. discoideum. A truncated form of PsA has been expressed in D. discoideum and secreted into a peptone based broth at levels of 10 mg per 1 growth medium. A simple purification protocol for recombinant PsA (rPsA) involved three steps: the concentration of the culture supernatant by ammonium sulfate precipitation, Mono Q anion-exchange chromatography, followed by size exclusion chromatography on Superdex 75. 20 mg of rPsA was purified to 98% purity from 37.1 culture supernatant. Purified rPsA was characterised. The molecular mass of the purified rPsA is 15.6 kDa, which suggests that the molecule is secreted as a monomer and contains 12% (w/w) carbohydrate. The protein sequence of rPsA proved identical to that of the predicted DNA construct. Although the recombinant form of PsA is expressed at a different developmental stage from the native molecule, the same Thr residues that are O-glycosylated in the authentic molecule are glycosylated in the recombinant protein.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antigens, Protozoan*
  • Antigens, Surface / biosynthesis*
  • Antigens, Surface / chemistry
  • Antigens, Surface / isolation & purification
  • Base Sequence
  • Blotting, Western
  • Chromatography, Gel
  • Chromatography, Ion Exchange
  • Cloning, Molecular
  • DNA Primers
  • Dictyostelium / genetics
  • Dictyostelium / metabolism*
  • Electrophoresis, Polyacrylamide Gel
  • Fungal Proteins / biosynthesis*
  • Fungal Proteins / chemistry
  • Fungal Proteins / isolation & purification
  • Genetic Vectors
  • Glycosylphosphatidylinositols / metabolism
  • Membrane Glycoproteins / biosynthesis*
  • Membrane Glycoproteins / chemistry
  • Membrane Glycoproteins / isolation & purification
  • Molecular Sequence Data
  • Peptide Fragments / chemistry
  • Peptide Fragments / isolation & purification
  • Plasmids
  • Polymerase Chain Reaction
  • Protein Folding
  • Protein Processing, Post-Translational
  • Protozoan Proteins*
  • Recombinant Proteins / biosynthesis*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Restriction Mapping

Substances

  • Antigens, Protozoan
  • Antigens, Surface
  • DNA Primers
  • Fungal Proteins
  • Glycosylphosphatidylinositols
  • Membrane Glycoproteins
  • Peptide Fragments
  • Protozoan Proteins
  • Recombinant Proteins
  • prespore-specific antigen, Dictyostelium discoideum