Large-scale preparation of recombinant platelet-derived growth factor AA secreted from recombinant baby hamster kidney cells.
The short isoform of platelet-derived growth factor A (PDGF-A) was expressed in a mammalian host (BHK-21 cell). A cell line was obtained that secreted up to 0.3 micrograms/10(6) cells recombinant PDGF-A chain homodimer/day into the medium. For large-scale production of supernatant, cells were grown either in roller bottles or in 2.5-1 stirred tank fermenters. A simple two-step procedure was developed to purify recombinant PDGF-AA (rPDGF-AA). The first step was adsorption onto porous glass and the final step was reversed-phase high-performance liquid chromatography. The yield was 0.2 mg/l supernatant. A total amount of 20-30 mg pure rPDGF-AA may be obtained from a single fermenter run. Sequence analysis showed the correct amino terminus and no internal proteolytic cleavages. The specific activity was 5 ng/ml for mouse AKR-2B cells. [125I]rPDGF-AA had an affinity constant of approximately 0.5 nM to these cells and 25,000 binding sites were estimated/cell.
Eichner
W
W
Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, Federal Republic of Germany.
Jäger
V
V
Herbst
D
D
Hauser
H
H
Hoppe
J
J
D000818:Animals; D002478:Cells, Cultured; D006224:Cricetinae; D007668:Kidney / Q000378:metabolism; D010982:Platelet-Derived Growth Factor / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis; D014162:Transfection
D010982:Platelet-Derived Growth Factor; D011994:Recombinant Proteins