Efficient production of biologically active human recombinant proteins in human lymphoblastoid cells from integrative and episomal expression vectors.
The ability of human lymphoblastoid cells to secrete large amounts of biologically active human hematopoietic growth factors from adenovirus-based expression vectors was investigated. The gene for human erythropoietin (EPO) was inserted into integrative (pTS39) and episomal (pTS53) vectors. Cell clones, originating from pTS39 or pTS53-transfected and stably selected cells, secreted recombinant human EPO (re-hEPO) at similar levels. The highest production, 60 mu/10(6) cells per 24 h, was obtained from a subclone of pTS39-transfected cells, grown in nonselective medium. The re-hEPO was shown to be biologically active in vivo by incorporation of 59Fe into red blood cells of polycythemic mice and in vitro by the proliferative response of the EPO-dependent cell line UT7. The purified protein of 36 kDa in SDS-PAGE slightly differed from re-hEPO from CHO cells. pTS39 vector was integrated at 15-30 copies per genome, whereas the pTS53 vector replicated at 10 copies per cell. Genes encoding human interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were also expressed in the integrative system as biologically active growth factors, demonstrating that our host-vector system allows the expression of any little gene or cDNA and efficient secretion of the re-protein produced.
Institut National de Transfusion Sanguine, Paris, France.
C
C
Lopez
A
A
de Chesnay
C
C
Tournamille
A
A
Ben Ghanem
S
S
Prigent
X
X
Drouet
P
P
Lambin
J P
JP
Cartron
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D004274:DNA, Recombinant; D004591:Electrophoresis, Polyacrylamide Gel; D004921:Erythropoietin / Q000235:genetics* / Q000378:metabolism; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007377:Interleukin-3 / Q000235:genetics* / Q000378:metabolism; D008214:Lymphocytes / Q000378:metabolism*; D051379:Mice; D008811:Mice, Inbred DBA; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D004274:DNA, Recombinant; D007377:Interleukin-3; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D004921:Erythropoietin; D016178:Granulocyte-Macrophage Colony-Stimulating Factor