title: Development of a Mammalian suspension culture for expression of active recombinant human urokinase-type plasminogen activator.
abstract: The development of specific catalytic inhibitors for the serine protease urokinase-type plasminogen activator (uPA) has been hindered due to difficulties in producing sufficient amounts of active recombinant uPA that is catalytically equivalent to native uPA. The purpose of this study was to develop an efficient system for the expression of recombinant human uPA that exhibits comparable proteolytic activity to that of the native protein. Since post-translational modifications (e.g. glycosylations) of uPA are necessary for efficient proteolytic activity, we have used a mammalian cell line [Chinese hamster ovary (CHO)-S] to express recombinant human uPA. CHO-S cells were selected to stably express full-length recombinant human uPA containing a hexahistidine tag at its C-terminus to permit purification by nickel-based affinity chromatography. Secretion of recombinant uPA into the culture media was confirmed by immunoblotting and the presence of an N-linked glycosylation was confirmed by PNGase sensitivity. Enzymatic activity of purified recombinant uPA was demonstrated using zymography and quantitatively compared to native uPA by kinetic analysis using an uPA-specific substrate. Native uPA and the recombinant uPA demonstrated comparable K(m) values (55.7 and 39 muM, respectively). Furthermore, inhibition studies using benzamidine resulted in a K(i) of 195 muM for native uPA, while recombinant uPA had a K(i) of 112 muM. These data indicate that recombinant human uPA expressed by CHO-S cells is functionally comparable to native uPA.
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Medicine, University of New Mexico, MSC08 4670, Albuquerque, NM, 87131-0001, USA.
authors.forename: Erica
authors.initials: E
authors.lastname: Atkins
authors.forename: Stephanie
authors.initials: S
authors.lastname: Zamora
authors.forename: Britny J
authors.initials: BJ
authors.lastname: Candia
authors.forename: Amy
authors.initials: A
authors.lastname: Baca
authors.forename: Robert A
authors.initials: RA
authors.lastname: Orlando