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  • Title: Expression of human tissue-type plasminogen activator from lytic viral vectors and in established cell lines.
    Author: Sambrook J, Hanahan D, Rodgers L, Gething MJ.
    Journal: Mol Biol Med; 1986 Dec; 3(6):459-81. PubMed ID: 3039288.
    Abstract:
    We have used two kinds of vectors to express a cDNA of human tissue plasminogen activator (t-PA) in mammalian cells. In one case, cDNAs inserted into vectors based on bovine papilloma virus were introduced into cultured murine cells and cell lines were established that efficiently and continuously secrete enzymatically active t-PA into the medium. Second, the t-PA gene was used to replace the sequences of the simian virus (SV40) genome that code for the viral coat proteins. Virus stocks were generated and used to infect a stable line of cultured simian cells. During the resulting lytic infection, expression of the t-PA gene is governed by the potent SV40 late promoter and enzymatically active t-PA accumulates rapidly in the medium. We have used these two vector systems to analyze the biosynthesis and transport of recombinant t-PA and to compare its properties with those of "natural" t-PA secreted by the Bowes line of human melanoma cells. t-PA secreted from all three sources is identical in specific activity (approximately 20,000 units/mg) despite differences in patterns of terminal glycosylation. Furthermore, non-glycosylated t-PA synthesized in the presence of tunicamycin was secreted efficiently and was indistinguishable in specific activity from glycosylated t-PAs.
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