Large scale purification and molecular characterization of human recombinant α1-proteinase inhibitor produced in yeasts
| Autor: | Alex Bollen, Anne Weyens, R. Loriau, Marc Hoylaerts, Piet Roelants, Michel De Wilde, Philippe Verdonck, P. Chuchana |
|---|---|
| Rok vydání: | 1987 |
| Předmět: |
Circular dichroism
biology Saccharomyces cerevisiae Bioengineering General Medicine biology.organism_classification Applied Microbiology and Biotechnology Molecular biology Yeast In vitro law.invention Biochemistry Affinity chromatography Polyclonal antibodies law biology.protein Recombinant DNA Pancreatic elastase Biotechnology |
| Zdroj: | Journal of Biotechnology. 5:181-197 |
| ISSN: | 0168-1656 |
| DOI: | 10.1016/0168-1656(87)90015-0 |
| Popis: | Human α 1 -proteinase inhibitor ( α 1 -PI), produced in yeast under the control of the yeast ARG3 promoter, was purified to homogeneity, following a multistep procedure that enables treatment of large quantities of yeast. The recombinant inhibitor, produced as a mature protein, is unglycosylated and has an apparent molecular weight (MW app ) of 44 000, in contrast to native glycosylated α 1 -PI which has a MW app of 54 000. Spectral analysis involving circular dichroism reveals a similar conformation for both recombinant and native α 1 -PI. Immunological comparison of both types of inhibitor showed complete immunological identity towards polyclonal antisera raised against the native homologue, indicating the total presence of antigenic determinants in the recombinant molecule. Recombinant α 1 -PI, as compared to its native counterpart, displays identical stability at acidic and alkaline pH; in addition, unfolding by urea occurs exactly with the same rate for both types of molecules. In vitro kinetic analysis of the inactivation rates of porcine pancreatic elastase and of bovine trypsin by recombinant and native α 1 -PI, respectively, reveal very similar second-order rate constants for both types of inhibitor indicating an intact reactive centre for the recombinant molecule. Provided that suitable administration schemes are developed, recombinant unglycosylated α 1 -proteinase inhibitor, purified on a large scale from yeast, may therefore find adequate therapeutic applications, e.g. in the treatment of emphysema or of local inflammation. |
| Databáze: | OpenAIRE |
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