Production of homogeneous basic fibroblast growth factor by specific enzymatic hydrolysis of larger microheterogeneous molecular forms
| Autor: | Gilles Cauet, F. Bertolero, Paolo Sarmientos, D. Betbeder, Pierre Monsan, P Caccia, G. Mazue, F. Paul, G Nitti |
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| Rok vydání: | 1991 |
| Předmět: |
Proteolysis
Molecular Sequence Data Basic fibroblast growth factor Bioengineering Applied Microbiology and Biotechnology Chromatography Affinity law.invention chemistry.chemical_compound Hydrolysis Residue (chemistry) Affinity chromatography law Enzymatic hydrolysis medicine Peptide bond Amino Acid Sequence medicine.diagnostic_test General Medicine Recombinant Proteins chemistry Biochemistry Recombinant DNA Biological Assay Fibroblast Growth Factor 2 Biotechnology |
| Zdroj: | Journal of Biotechnology. 21:83-92 |
| ISSN: | 0168-1656 |
| DOI: | 10.1016/0168-1656(91)90262-t |
| Popis: | The 146-amino acid form of basic fibroblast growth factor (bFGF) was expressed in Escherichia coli and purified by a two step process including ion exchange and heparin-Sepharose chromatographies. However, the resulting protein consisted of a mixture of 146- and 145-amino acid forms, indicating that, besides the initial methionine, also the following residue (proline) was removed from the N-terminus. The same phenomenon was observed when the 155-amino acid form, which is biologically equivalent to the shorter one, was expressed in E. coli. Taking into account the previously known data concerning the possible mechanism of cleavage of the extended forms of bFGF in vivo, we developed an efficient enzymatic process that allows the production of an homogeneous 146-amino acid form from recombinant NH2-end extended forms. This process takes advantage of the protecting effect that heparin exerts on bFGF. Accordingly, when bFGF, complexed to heparin, is treated with pepsin A, an aspartic protease with a broad specificity, only the Leu9-Pro10 peptide bond is cleaved generating the 146-amino acid form. Quantitative yields of this reaction are also achieved when bFGF is bound to a heparin-Sepharose column, allowing the integration of this enzymatic step directly during purification of the recombinant extended forms of bFGF. |
| Databáze: | OpenAIRE |
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