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. 2005 Sep;49(1):25–37. doi: 10.1007/s10616-005-4637-7

Development of a Mammalian Suspension Culture for Expression of Active Recombinant Human Urokinase-type Plasminogen Activator

Erica Atkins 1, Stephanie Zamora 1, Britny J Candia 1, Amy Baca 1, Robert A Orlando 1,✉
PMCID: PMC3449752  PMID: 19003060

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 Km values (55.7 and 39 μM, respectively). Furthermore, inhibition studies using benzamidine resulted in a Ki of 195 μM for native uPA, while recombinant uPA had a Ki of 112 μM. These data indicate that recombinant human uPA expressed by CHO-S cells is functionally comparable to native uPA.

Keywords: CHO-S, plasminogen, recombinant expression, serine protease, tumor cell motility, uPA, urokinase

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Glossary

CHO-S

Chinese hamster ovary-S cells

PAI-1

type-1 plasminogen activator inhibitor

PNGase F

peptide-N-glycosidase F

SDS-PAGE

sodium dodecylsulfate-polyacrylamide gel electrophoresis

uPA

urokinase-type plasminogen activator

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