Stabile production of a thrombin resistant pro-urokinase derivative (PRO-UKS1) by Namalwa KJM-1 cells adapted to serum-free medium.
Pro-UKS1 was designed as a thrombin-resistant derivative of pro-urokinase (pro-UK) by introducing a glycosylation site using site-directed mutagenesis. An expression plasmid for pro-UKS1, pMo1UKS1SEd1-5, was constructed and introduced into Namalwa KJM-1 cells (Hosoi et al., 1988), and cells resistant to G418 and Methotrexate (MTX) were obtained. Amongst them, the highest pro-UKS1 producer (resistant to 500 nM of MTX), clone 41-8, was selected and further characterized. Clone 41-8 was cultured in serum-free ITPSGF medium (Hosoi et al., 1988). Under the conventional conditions, the concentration of pro-UKS1 reached 26 micrograms ml-1. Addition of glucose and tri-iodothyronine (T3) improved productivity, and the maximal productivity of pro-UKS1 was 67 micrograms ml-1 day-1. In this conditioned medium, content of pro-UKS1 was above 80% of total proteins.
Hosoi
S
S
Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., Japan.
Satoh
M
M
Miyaji
H
H
Nishi
T
T
Mizukami
T
T
Hasegawa
M
M
Itoh
S
S
Tamaoki
T
T
D000595:Amino Acid Sequence; D002051:Burkitt Lymphoma / Q000473:pathology*; D017077:Culture Media, Conditioned / Q000494:pharmacology; D016895:Culture Media, Serum-Free; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005947:Glucose / Q000494:pharmacology; D006031:Glycosylation; D006801:Humans; D008727:Methotrexate / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000187:drug effects / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification; D012641:Selection, Genetic; D013917:Thrombin / Q000494:pharmacology*; D014162:Transfection; D014284:Triiodothyronine / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000187:drug effects; D014568:Urokinase-Type Plasminogen Activator / Q000096:biosynthesis* / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification
D017077:Culture Media, Conditioned; D016895:Culture Media, Serum-Free; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014284:Triiodothyronine; D013917:Thrombin; D014568:Urokinase-Type Plasminogen Activator; D005947:Glucose; C067551:saruplase; D008727:Methotrexate