A high-level and regulatable production system for recombinant glycoproteins using a human interferon-alpha promoter-based expression vector.
A novel expression vector for human cells, pIFP, which expresses a cloned gene under the control of the human interferon-alpha-encoding gene (IFN-alpha) promoter (pIFN) was constructed. As a model of glycoprotein production, a human erythropoietin-encoding cDNA (EPO) inserted downstream from pIFN in pIFP was introduced into human B-cell leukemia-derived BALL-1 cells, and EPO-producing cells were established. Upon stimulation with Sendai virus, the cells produced human EPO at high levels. The highest production level and the highest inducibility were 872 IU/ml and 67-fold, respectively. Simultaneously, the transformed cells also produced IFN-alpha and tumor necrosis factor-alpha (TNF-alpha), as the parental BALL-1 cells did. Comparing the amounts of the substances produced, activity of the exogenous pIFN introduced seemed much higher than that of the endogenous one. Further, the transformed cells could be obtained in a large quantity by being applied to the 'in vivo cell proliferation method (hamster method)'. Human EPO produced by the transformed cells had a molecular mass range of 35 to 42 kDa, similar to that of EPO produced by CHO cells. The processing of EPO seemed to occur properly. The combination of the human pIFN, BALL-1 cells and the hamster method provides us with a useful production system for bioactive glycoproteins of human origin.
Fujisaki Institute, Hayashibara Biochemical Laboratories, Inc., Okayama, Japan.
T
T
Mori
K
K
Yamamoto
T
T
Ohta
C
C
Sakamoto
M
M
Sato
K
K
Koide
T
T
Murakami
M
M
Fujii
S
S
Fukuda
M
M
Kurimoto
D000818:Animals; D006224:Cricetinae; D018076:DNA, Complementary; D004921:Erythropoietin / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D005822:Genetic Vectors; D006023:Glycoproteins / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D016898:Interferon-alpha / Q000096:biosynthesis / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014170:Transformation, Genetic; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000235:genetics
D018076:DNA, Complementary; D006023:Glycoproteins; D016898:Interferon-alpha; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D004921:Erythropoietin