Plasminogen activator inhibitor type 2 cDNA transfected into Chinese hamster ovary cells is stably expressed but not secreted |
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| Affiliation: | 1. National Key Laboratory for Precision Hot Processing of Metals, Harbin Institute of Technology, Harbin, 150001, PR China;2. School of Materials Science and Engineering, Harbin Institute of Technology, Harbin, 150001, PR China;3. School of Materials Science and Engineering, Heilongjiang University of Science and Technology, Harbin, 150022, PR China;4. School of Materials Science and Engineering, Jiangsu University of Science and Technology, Zhenjiang, 212003, PR China;1. State Key Laboratory of Chemical Engineering, East China University of Science and Technology, Shanghai, 200237, China;2. Engineering Research Center of Advanced Functional Material Manufacturing of Ministry of Education, Zhengzhou University, Zhengzhou, 450001, China;1. Université Lille Nord de France, faculté de médecine, 59000 Lille, France;2. CHRU de Lille, hôpital Claude-Huriez, service de médecine interne, centre national de référence de la sclérodermie systémique, 59037 Lille cedex, France;3. EA2686, 59045 Lille, France;4. Université Paris-Sud, faculté de médecine, 94270 Le Kremlin-Bicêtre, France;5. AP–HP, hôpital Bicêtre, service de pneumologie, DHU Thorax Innovation, 94275 Le Kremlin-Bicêtre, France;6. Inserm U999, Centre chirurgical Marie-Lannelongue, LabEx LERMIT, 92350 Le Plessis-Robinson, France;7. AP–HP, université Paris-Descartes, hôpital Cochin, service de médecine interne, Centre national de référence des vascularites nécrosantes et de la sclérodermie systémique, 75014 Paris, France;8. Centre chirurgical Marie-Lannelongue, service de chirurgie thoracique, vasculaire et de transplantation cardiopulmonaire, 92350 Le Plessis-Robinson, France;9. AP–HP, hôpital Bicêtre, service de chirurgie générale et digestive, 94275 Le Kremlin-Bicêtre, France;10. AP–HP, université Paris-Descartes, hôpital Cochin, service de gastroentérologie, 75014 Paris, France;11. Centre chirurgical Marie-Lannelongue, service de réanimation adulte, 92350 Le Plessis-Robinson, France;12. CHRU de Lille, hôpital cardiologique, service de cardiologie, 59034 Lille cedex, France;13. AP–HP, université Paris-Descartes, hôpital Cochin, service de radiologie, 75014 Paris, France;14. Université Claude-Bernard Lyon 1, hôpital Louis-Pradel, service de pneumologie, Centre de référence national des maladies pulmonaires rares, 69000 Lyon, France;15. AP–HP, hôpital Saint-Louis, service de médecine interne et pathologie vasculaire, 75010 Paris, France;16. CHRU de Lille, hôpital Calmette, service de pneumologie et immunoallergologie, clinique des maladies respiratoires, Centre de compétence maladies pulmonaires rares, 59000 Lille, France;1. Institute of Nutrition, Friedrich Schiller University Jena, 07743 Jena, Germany;2. Department of Molecular Hemostaseology, Jena University Hospital, 07743 Jena, Germany;3. Integrated Research and Treatment Center, Center for Sepsis Control and Care (CSCC), Jena University Hospital, 07747 Jena, Germany |
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| Abstract: | ![]()
Immunological screening with a monoclonal antibody probe developed against the plasminogen activator inhibitor type 2 (PAI-2) detected 6 positive clones from approximately 1.7 × 105 recombinant phages in a λgt11 expression library containing cDNA inserts prepared from human placental mRNA. Hybridisation experiments at high stringency indicated that the 6 clones were related. One positive clone was found to produce a fusion protein of Mr 170 000 that was recognised by both monoclonal and polyclonal antibodies against PAI-2. In order to obtain a full length cDNA clone for PAI-2, an additional cDNA library was screened. From this screening, we obtained a cDNA clone that encoded all but a portion of the 5′-untranslated region of the PAI-2 mRNA. Primer extension experiments determined that the 5'-untranslated region was 74 nucleotides in length. The PAI-2 mRNA has an open reading frame of 1245 nucleotides and encodes a 46,6 kDa protein. Analysis of the predicted amino acid sequence revealed that like ovalbumin, PAI-2 has an internal non-cleaved signal peptide. The PAI-2 coding sequence is followed by a 3'-untranslated region of 581 nucleotides. In order to study secretion of PAI-2, a plasmid construct containing the PAI-2 cDNA preceded by the SV40 early promotor was transfected into Chinese hamster ovary cells. The PAI-2 cDNA was efficiently expressed in these cells but unexpectedly the protein was not secreted into the culture medium. The absence of PAI-2 secretion in Chinese hamster ovary cells may be due to the lack of a ‘tissue specific secretory mechanism’ that is present in tissues which normally express PAI-2 |
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