Expression and purification of a recombinant buforin derivative from Escherichia coli
| Autor: | Jin-Suk Cho, Hong-Rak Kim, Byung-Hee Han, Jae-Hyun Lee, Heung-Bok Park, Yeon-Sung Park, Sang-Hyun Pyo |
|---|---|
| Rok vydání: | 2004 |
| Předmět: |
chemistry.chemical_classification
Chromatography Molecular mass Ion chromatography Bioengineering Peptide medicine.disease_cause Applied Microbiology and Biotechnology Biochemistry law.invention chemistry.chemical_compound Hydroxylamine chemistry law medicine Recombinant DNA Escherichia coli Ammonium sulfate precipitation Homogenization (biology) |
| Zdroj: | Process Biochemistry. 39:1731-1736 |
| ISSN: | 1359-5113 |
| DOI: | 10.1016/j.procbio.2003.07.007 |
| Popis: | An Escherichia coli expression system was constructed for production of a recombinant antimicrobial peptide buforin derivative named buforin IIb and a large-scale process of cultivation and purification was also developed. Inclusion bodies of fusion proteins containing recombinant buforin IIb were released from high-density-cultured cells by high-pressure homogenization. The inclusion bodies solubilized in a buffer containing 8 M urea were incubated in cleavage buffer containing 1.7 M hydroxylamine. The crude mixtures were fractionated by ammonium sulfate precipitation and then washed with distilled water to remove salts. The pellets were dissolved in 7 M urea solution, and treated with polyethyleneimine (PEI) to remove contaminated DNA. The recombinant buforin IIb was purified by cation exchange chromatography and reverse phase HPLC (RP-HPLC). The purity of the recombinant buforin IIb was determined by RP-HPLC and SDS-PAGE and the molecular mass of the recombinant buforin IIb analyzed by MALDI-TOF-MS showed good agreement with the authentic buforin IIb. These results suggest that the production method in this report is useful in obtaining a large quantity of recombinant antimicrobial peptide. |
| Databáze: | OpenAIRE |
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