Expression of human plasminogen in Drosophila Schneider S2 cells.
The cDNA that encodes full-length human plasminogen (Glu1-hPg) has been expressed in Drosophila Schneider S2 cells under the influence of the Drosophila BiP protein signal sequence, which allowed the protein to be secreted into the medium. A procedure was devised for clonal selection of high-expressing cells, which were then used for large-scale expression of 10-15 mg/liter of the protein in the culture medium. The protein produced using this system was extensively characterized and contained full-length recombinant (r) Glu1-hPg plasminogen. As with human plasma Glu1-hPg, the S2-expressed protein underwent the Cl--induced transition to the tight conformation, which resulted in a weakly activatable zymogen. The addition of the ligand, epsilon-amino caproic acid, induced the relaxed conformation of r-Glu1-hPg, which was highly activatable, again in agreement with similar data for human plasma Glu1-hPg. The thermal stability of the S2-expressed r-Glu1-hPg also correlated well with that of human plasma hPg. These studies show that intact r-Glu1-hPg can be produced in high yield in Drosophila Schneider S2 cells, which possesses similar properties to its human plasma counterpart.
Nilsen
S L
SL
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA.
Castellino
F J
FJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004330:Drosophila / Q000235:genetics; D004355:Drug Stability; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D010957:Plasmids / Q000235:genetics; D010958:Plasminogen / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics; D014162:Transfection
D017931:DNA Primers; D011994:Recombinant Proteins; D010958:Plasminogen