Expression, purification and characterisation of secreted recombinant glycoprotein PsA in Dictyostelium discoideum.
J Biotechnol
; 38(2): 137-49, 1995 Jan 15.
Article
en En
| MEDLINE
| ID: mdl-7765806
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.
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Banco de datos:
MEDLINE
Asunto principal:
Proteínas Recombinantes
/
Proteínas Fúngicas
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Glicoproteínas de Membrana
/
Proteínas Protozoarias
/
Dictyostelium
/
Antígenos de Protozoos
/
Antígenos de Superficie
Límite:
Animals
Idioma:
En
Año:
1995
Tipo del documento:
Article