title: Recombinant production of a hybrid plasminogen activator composed of surfactant protein B and low-molecular-weight urokinase.
abstract: 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 approximately 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.
authors.affiliation: Department of Internal Medicine, University of Giessen Lung Center, Giessen, Germany.
authors.forename: Clemens
authors.initials: C
authors.lastname: Ruppert
authors.forename: Poornima
authors.initials: P
authors.lastname: Mahavadi
authors.forename: Malgorzata
authors.initials: M
authors.lastname: Wygrecka
authors.forename: Timothy E
authors.initials: TE
authors.lastname: Weaver
authors.forename: Viktor
authors.initials: V
authors.lastname: Magdolen
authors.forename: Steven
authors.initials: S
authors.lastname: Idell
authors.forename: Klaus T
authors.initials: KT
authors.lastname: Preissner
authors.forename: Werner
authors.initials: W
authors.lastname: Seeger
authors.forename: Andreas
authors.initials: A
authors.lastname: Günther
authors.forename: Philipp
authors.initials: P
authors.lastname: Markart
mesh_terms: D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002364:Caseins / Q000378:metabolism; D002470:Cell Survival; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D005240:Feasibility Studies; D015870:Gene Expression; D006801:Humans; D008970:Molecular Weight; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism; D017434:Protein Structure, Tertiary; D037701:Pulmonary Surfactant-Associated Protein B / Q000096:biosynthesis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013997:Time Factors; D014162:Transfection; D014568:Urokinase-Type Plasminogen Activator / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics
chemical_list: D002364:Caseins; D017395:Plasminogen Activator Inhibitor 1; D037701:Pulmonary Surfactant-Associated Protein B; D011993:Recombinant Fusion Proteins; D014568:Urokinase-Type Plasminogen Activator