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

• Published in: Journal of biotechnology 1995-01, Vol.38 (2), p.137-149
• Main Authors: Zhou-Chou, Ti, Slade, Martin B., Williams, Keith L., Gooley, Andrew A.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Antigens, Protozoan; Antigens, Surface - biosynthesis; Antigens, Surface - chemistry; Antigens, Surface - isolation & purification; Base Sequence; Biological and medical sciences; Biotechnology; Blotting, Western; Chromatography, Gel; Chromatography, Ion Exchange; Cloning, Molecular; Dictyostelium - genetics; Dictyostelium - metabolism; Dictyostelium discoideum; DNA Primers; Electrophoresis, Polyacrylamide Gel; Eukaryote expression system; Fundamental and applied biological sciences. Psychology; Fungal Proteins - biosynthesis; Fungal Proteins - chemistry; Fungal Proteins - isolation & purification; Genetic engineering; Genetic technics; Genetic Vectors; Glycosylphosphatidylinositols - metabolism; Membrane Glycoproteins - biosynthesis; Membrane Glycoproteins - chemistry; Membrane Glycoproteins - isolation & purification; Methods. Procedures. Technologies; Modification of gene expression level; Molecular Sequence Data; Peptide Fragments - chemistry; Peptide Fragments - isolation & purification; Plasmids; Polymerase Chain Reaction; Protein Folding; Protein Processing, Post-Translational; Protozoan Proteins; Recombinant glycoprotein; Recombinant protein purification; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Restriction Mapping
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/0168-1656(94)00127-X

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 l 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 3.7 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.