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Updated: Sep 25, 2026

A Scalable, Cell-Based Method for the Functional Assessment of Ube3a Variants
Published on: October 10, 2022
Characterization of single chain urokinase-type plasminogen activator with a novel amino-acid substitution in the
M Yoshimoto1, Y Ushiyama, M Sakai
1Department of Applied Biology, Research Center, Taisho Pharmaceutical Co., Ltd., Saitama, Japan.
Abstract:
ECV304 is a cell line established by a spontaneous transformation of endothelial cells of a human umbilical vein. It was shown that ECV304 secretes single chain urokinase-type plasminogen activator (scu-PA). A subclone, ECV304 clone 15, was obtained by acclimatization of parental clone to serum-free medium followed by limiting dilution. The clone was found to produce approximately five times as much scu-PA (approximately 20 IU/10(6) cells per day) as the parental clone after a 40 days' culture. Though the biochemical characteristics of the purified scu-PA were indistinguishable from those of the native scu-PA, it had a lower affinity for fibrin clots under the employed conditions. Molecular cloning of a cDNA encoding the scu-PA has identified a novel substitution from C to T in the nucleotide sequence encoding the kringle structure. The substitution resulted in an alteration from Pro (CCG) to Leu (CTG) at amino-acid position 121, which may be directly or indirectly involved in the decrease in the apparent affinity.
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