TY - JOUR
T1 - Expression, purification and characterisation of secreted recombinant glycoprotein PsA in Dictyostelium discoideum
AU - Zhou-Chou, Ti
AU - Slade, Martin B.
AU - Williams, Keith L.
AU - Gooley, Andrew A.
PY - 1995/1/15
Y1 - 1995/1/15
N2 - 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.
AB - 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.
KW - Dictyostelium discoideum
KW - Eukaryote expression system
KW - Recombinant glycoprotein
KW - Recombinant protein purification
UR - http://www.scopus.com/inward/record.url?scp=0028894693&partnerID=8YFLogxK
U2 - 10.1016/0168-1656(94)00127-X
DO - 10.1016/0168-1656(94)00127-X
M3 - Article
C2 - 7765806
AN - SCOPUS:0028894693
SN - 0168-1656
VL - 38
SP - 137
EP - 149
JO - Journal of Biotechnology
JF - Journal of Biotechnology
IS - 2
ER -