Stabile production of a thrombin resistant pro-urokinase derivative (PRO-UKS1) by Namalwa KJM-1 cells adapted to serum-free medium

Cytotechnology. 1995;19(1):1-10. doi: 10.1007/BF00749750.

Abstract

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.

Publication types

  • Comparative Study

MeSH terms

  • Amino Acid Sequence
  • Burkitt Lymphoma / pathology*
  • Culture Media, Conditioned / pharmacology
  • Culture Media, Serum-Free
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme-Linked Immunosorbent Assay
  • Glucose / pharmacology
  • Glycosylation
  • Humans
  • Methotrexate / pharmacology
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Protein Processing, Post-Translational
  • Recombinant Fusion Proteins / biosynthesis*
  • Recombinant Fusion Proteins / drug effects
  • Recombinant Fusion Proteins / isolation & purification
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / drug effects
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Selection, Genetic
  • Thrombin / pharmacology*
  • Transfection
  • Triiodothyronine / pharmacology
  • Tumor Cells, Cultured / drug effects
  • Urokinase-Type Plasminogen Activator / biosynthesis*
  • Urokinase-Type Plasminogen Activator / drug effects
  • Urokinase-Type Plasminogen Activator / genetics
  • Urokinase-Type Plasminogen Activator / isolation & purification

Substances

  • Culture Media, Conditioned
  • Culture Media, Serum-Free
  • Recombinant Fusion Proteins
  • Recombinant Proteins
  • Triiodothyronine
  • Thrombin
  • Urokinase-Type Plasminogen Activator
  • Glucose
  • saruplase
  • Methotrexate