Synthesis and Purification of Active Human Tissue Plasminogen Activator From Escherichia coli
| Autor: | Fabienne Parker, Patrice Denefle, Terence Cartwright, Jean-Francois Mayaux, Nadine Delporte, Paolo Sarmientos, Marc Duchesne, J. Boiziau, Nadine Fromage, Yves Lelièvre |
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| Rok vydání: | 1989 |
| Předmět: |
Signal peptide
Glycosylation Lysis integumentary system biology Biomedical Engineering Bioengineering Periplasmic space biology.organism_classification medicine.disease_cause Applied Microbiology and Biotechnology Tissue plasminogen activator Enterobacteriaceae Molecular biology chemistry.chemical_compound Biochemistry chemistry medicine Molecular Medicine Escherichia coli Plasminogen activator Biotechnology medicine.drug |
| Zdroj: | Nature Biotechnology. 7:495-501 |
| ISSN: | 1546-1696 1087-0156 |
| DOI: | 10.1038/nbt0589-495 |
| Popis: | Using different types of efficient Escherichia coli expression systems, we have been able to obtain levels of human tissue-type plasminogen activator (tPA) reaching at least five percent of total bacterial proteins. The natural precursor of tPA (pre-tPA) expressed in E. coli accumulates, most probably in the cytoplasm, partly due to the low efficiency of the tPA leader sequence as a signal for secretion into the E. coli periplasm. We also expressed mature tPA in an insoluble, aggregated form and treatments were developed that permitted significant renaturation of this protein from the lysis pellet. Further characterization and purification of the rena-tured tPA polypeptide strongly suggest that a fully active enzyme, very similar to the eukaryotic product can be obtained, despite the total lack of glycosylation. |
| Databáze: | OpenAIRE |
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