Constitutive high-level production of human lymphotoxin by CHO-K1 cells transformed with the human lymphotoxin gene controlled by a human beta-actin promoter.
To achieve high-level production of human lymphotoxin (hLT), a plasmid (p beta LT-ldhfr) containing the hLT genomic DNA, a mouse dihydrofolate reductase (DHFR) cDNA, and a bacterial Ecogpt gene was cotransfected with a plasmid (p beta LTML) encoding only the hLT genomic DNA into Chinese hamster ovary (CHO-K1) cells at a 1:7 molar ratio. Subsequently one of the Ecogpt-positive clones (clone A31) was grown in stepwise increasing concentrations of methotrexate (MTX). A large amount of the hLT was secreted by cells resistant to increased levels of MTX as a result of coamplification of the DHFR cDNA and the hLT gene. A cell clone (clone M-1) resistant to up to 500 nM MTX constitutively expressed the hLT at a concentration of 80 micrograms per ml at an elevated level for about 2 months. The hLT was produced in glycosylated form the molecular mass of which was 23,000 daltons and the mRNA was normally spliced, so the protein molecules were probably homogeneous.
Nakagawa
K
K
Biochemical Research Laboratories, Kanegafuchi Chemical Industry Co., Ltd., Hyogo, Japan.
Yajima
K
K
Yamashita
K
K
Ikenaka
Y
Y
Yokota
S
S
Kakutani
T
T
Kawaharada
H
H
Watanabe
K
K
D000199:Actins / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D006224:Cricetinae; D004247:DNA / Q000235:genetics; D005260:Female; D005784:Gene Amplification; D006801:Humans; D008233:Lymphotoxin-alpha / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012091:Repetitive Sequences, Nucleic Acid; D013539:Simian virus 40 / Q000235:genetics; D014170:Transformation, Genetic
D000199:Actins; D008233:Lymphotoxin-alpha; D004247:DNA