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.
Biochemical Research Laboratories, Kanegafuchi Chemical Industry Co., Ltd., Hyogo, Japan.
K
K
Nakagawa
K
K
Yajima
K
K
Yamashita
Y
Y
Ikenaka
S
S
Yokota
T
T
Kakutani
H
H
Kawaharada
K
K
Watanabe
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