Expression, purification and characterisation of secreted recombinant glycoprotein PsA in Dictyostelium discoideum.
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.
Slade
M B
MB
Macquarie University Centre for Analytical Biotechnology, School of Biological Sciences, Macquarie University, Sydney, NSW, Australia.
Williams
K L
KL
Gooley
A A
AA
D000595:Amino Acid Sequence; D000818:Animals; D000953:Antigens, Protozoan; D000954:Antigens, Surface / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D001483:Base Sequence; D015153:Blotting, Western; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D017931:DNA Primers; D004023:Dictyostelium / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D005822:Genetic Vectors; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010957:Plasmids; D016133:Polymerase Chain Reaction; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D015800:Protozoan Proteins; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D015183:Restriction Mapping
D000953:Antigens, Protozoan; D000954:Antigens, Surface; D017931:DNA Primers; D005656:Fungal Proteins; D017261:Glycosylphosphatidylinositols; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D015800:Protozoan Proteins; D011994:Recombinant Proteins; C074496:prespore-specific antigen, Dictyostelium discoideum