Recombinant production of a hybrid plasminogen activator composed of surfactant protein B and low-molecular-weight urokinase

Clemens Ruppert; Poornima Mahavadi; Malgorzata Wygrecka; Timothy E. Weaver; Viktor Magdolen; Steven Idell; Klaus T. Preissner; Werner Seeger; Andreas Günther; Philipp Markart

doi:10.1160/TH08-03-0142

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2008; 100(06): 1185-1192
DOI: 10.1160/TH08-03-0142

New Technologies, Diagnostic Tools and Drugs

Schattauer GmbH

Recombinant production of a hybrid plasminogen activator composed of surfactant protein B and low-molecular-weight urokinase

Clemens Ruppert

¹Department of Internal Medicine, University of Giessen Lung Center (UGLC), Giessen, Germany

,

Poornima Mahavadi

¹Department of Internal Medicine, University of Giessen Lung Center (UGLC), Giessen, Germany

,

Malgorzata Wygrecka

²Department of Biochemistry, University of Giessen Lung Center (UGLC), Giessen, Germany

,

Timothy E. Weaver

³Division of Pulmonary Biology, Childrens Hospital Medical Center, Cincinnati, Ohio, USA

,

Viktor Magdolen

⁴Department of Gynecology, Klinikum Rechts der Isar, TU Munich, Germany

,

Steven Idell

⁵Department of Medical Specialties, The University of Texas Health Center at Tyler, Texas, USA

,

Klaus T. Preissner

²Department of Biochemistry, University of Giessen Lung Center (UGLC), Giessen, Germany

,

Werner Seeger

¹Department of Internal Medicine, University of Giessen Lung Center (UGLC), Giessen, Germany

,

Andreas Günther^*

¹Department of Internal Medicine, University of Giessen Lung Center (UGLC), Giessen, Germany

,

Philipp Markart^*

¹Department of Internal Medicine, University of Giessen Lung Center (UGLC), Giessen, Germany

› Author Affiliations

Abstract

Summary

Intraalveolar fibrin deposition is commonly observed during acute inflammatory and chronic interstitial lung diseases and may contribute to impairment of surfactant function and gas exchange.We recently described a chemically cross-linked chimeric protein consisting of surfactant protein (SP)-B and urokinase (uPA) for targeting alveolar fibrin under conditions such as acute respiratory distress syndrome (ARDS) or lung fibrosis.We now investigated the feasibility of a recombinant production of a fusion protein encoding mature SP-B and uPA, termed SPUC. Four different SPUC proteins (N-term SP-B/C-term uPA, N-term uPA/C-term SP-B, each +/− His-tag) were prepared by cloning the cDNA encoding mature SP-B and low-molecular-weight uPA into the expression vector pcDNA3.1. CHO-cells were transfected with the constructs and the supernatant and cell lysates were analyzed for expression of SPUC.Using a chromogenic substrate assay uPA activity was found in supernatants and lysates of transfected cells with highest activities related to the N-term uPA/C-term SP-B (+/− His-tag) construct in supernatants 48h after transfection.Casein enzymography showed an enzymatically active fusion proteins with a molecular weight of ∼ 42 kDa in the supernatant of cells transfected with the N-term uPA/C-term SP-B (+/− His-tag) construct,but only a minor activity with the N-term SP-B/C-term uPA construct. The N-term uPA/C-term SP-B construct was also shown to possess higher resistance towards inhibition by plasminogen activator inhibitor-1.We conclude that recombinant production of a fusion protein consisting of mature SP-B and uPA is feasible, when the SP-B moiety is fused to the C-terminus of urokinase.

Keywords

Urokinase - pulmonary surfactant - acute lung injury - fusion protein

Full Text

References

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