The role of carbohydrate in the function of human plasminogen: comparison of the protein obtained from molecular cloning and expression in Escherichia coli and COS cells.
A cDNA library was constructed in the phage lambda gt11 from human liver mRNA enriched for plasminogen mRNA by chromatography on Sepharose 4B. A full-length cDNA clone of human plasminogen was isolated. The 2.7 kb cDNA encoded the entire plasminogen molecule, a signal peptide sequence and two start codons with a 5'-untranslated region of about 80 base pairs. In the 3'-non coding region of 280 base pairs a consensus signal AATAAA was found at a distance of 46 base pairs upstream of the poly(A) tail. The plasminogen cDNA was subcloned in the eukaryotic expression vector p91023 (B), and human plasminogen was expressed in monkey kidney (COS m6) cells and in Escherichia coli. The recombinant molecule obtained from COS cells has physicochemical and biological properties similar to native human plasminogen I, indicating that it has folded in a manner similar to plasminogen synthesized by liver. By contrast, plasminogen expressed in E. coli could not be activated and showed biological properties which are very different from glycosylated forms of plasminogen. However, the non-glycosylated plasminogen was bound by lysine-Sepharose and reacted with a conformation dependent monoclonal antibody to kringles 1 to 3. These data suggest that the protein has properly folded kringle domains. Our studies suggest that the carbohydrate domains may play an important role in the function of the plasminogen molecule.
Department of Pathology, Duke University Medical Center, Durham, NC 27710.
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Gonzalez-Gronow
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Grenett
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Fuller
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Pizzo
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D015723:Gene Library; D006023:Glycoproteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D051379:Mice; D010958:Plasminogen / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism / Q000493:pharmacokinetics; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000493:pharmacokinetics; D014018:Tissue Distribution; D014162:Transfection
D006023:Glycoproteins; D011994:Recombinant Proteins; D010958:Plasminogen; D004247:DNA