High Yield Expression and Purification of Human Endothelin-1
| Autor: | Silvia Merli, S. Germani, Giovanni Cassani, Giorgio Fassina, Gennaro Ciliberto |
|---|---|
| Rok vydání: | 1994 |
| Předmět: |
medicine.hormone
Monosaccharide Transport Proteins Recombinant Fusion Proteins medicine.medical_treatment Molecular Sequence Data Polymerase Chain Reaction Chromatography Affinity Maltose-Binding Proteins law.invention Endothelins Maltose-binding protein Affinity chromatography law Endopeptidases Escherichia coli medicine Chymotrypsin Humans Amino Acid Sequence Cloning Molecular Protein Precursors Chromatography High Pressure Liquid Protease Chromatography Base Sequence Endothelin-1 biology Chemistry Escherichia coli Proteins Trypsin Endothelin 1 Biochemistry Recombinant DNA biology.protein ATP-Binding Cassette Transporters Carrier Proteins Biotechnology medicine.drug |
| Zdroj: | Protein Expression and Purification. 5:559-568 |
| ISSN: | 1046-5928 |
| DOI: | 10.1006/prep.1994.1077 |
| Popis: | A DNA construct encoding human big endothelin (Big ET) preceded by the factor Xa protease recognition site (Ile-Glu-Gly-Arg), fused in frame to the maltose binding protein sequence, has been introduced in DH5-alpha cells. The fusion product (MBP-Big ET) was expressed at a concentration close to 100 micrograms/ml of culture broth and constituted approximately 50% of the total protein content. Crude cell extracts containing the fusion product have been directly treated with trypsin under mild denaturing conditions in order to release big endothelin (1-37) from the adduct. Cleavage yield of the MBP-Big ET adduct was close to 70%. Big ET(1-37) was separated from unrelated peptides derived from the tryptic digest of the bacterial extract by affinity chromatography. The affinity column was prepared by immobilizing a protease resistant peptide ligand able to recognize Big ET with sufficient affinity, selectivity, and specificity. From the affinity step (recovery, 90%), recombinant Big ET(1-37) was obtained with a purity close to 80%. The affinity-purified recombinant product was then digested with alpha-chymotrypsin in order to release endothelin (1-21), which was then purified by RP-HPLC. With this two-step purification protocol, 3 micrograms of endothelin was recovered from 1 ml of bacterial broth, with a purity close to 95%. |
| Databáze: | OpenAIRE |
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