Expressing and purifying an anti-atherosclerosis polypeptide vaccine in Escherichia coli
| Autor: | Qi Gaofu, Liu Jing-jing, Wu Jie, Yang Xin, Rouel S. Roque |
|---|---|
| Rok vydání: | 2004 |
| Předmět: |
Proline
Arteriosclerosis Recombinant Fusion Proteins Molecular Sequence Data Peptide medicine.disease_cause Mice chemistry.chemical_compound Hydrolysis Bacterial Proteins Tetanus Toxin Western blot Aspartic acid Escherichia coli medicine Animals Humans Amino Acid Sequence Glycoproteins Inclusion Bodies chemistry.chemical_classification Aspartic Acid Chromatography medicine.diagnostic_test Chemistry Fusion protein Cholesterol Ester Transfer Proteins Biochemistry Antibody Formation Vaccines Subunit Urea Immunization Hydrochloric Acid Carrier Proteins Glycoprotein Dimerization Biotechnology |
| Zdroj: | Protein Expression and Purification. 36:198-206 |
| ISSN: | 1046-5928 |
| Popis: | A chimeric polypeptide of TTP-CETPC was successfully expressed as inclusion bodies in Escherichia coli by fusing it with the C-terminus of asparaginase and a basic amino acid-rich peptide (KR). After partially purified by washing with 0.5% (v/v) Triton X-100 in 10 mM PB, the pellet was solubilized in 8 M urea. The solution was precipitated with single volume and double volumes of cold ethanol for removing impurities. The fusion protein in solution was precipitated with triple volumes of ethanol to increase purity and then hydrolyzed with 50 mM hydrochloric acid at 55 degrees C for 72 h. The TTP-CETPC polypeptide was released after the unique acid-labile aspartylprolyl bond in the fusion protein was cleaved by acid. After impurities were removed by adjusting the hydrolysis solution pH to 9.45 and then to 8.37, the TTP-CETPC polypeptide was further purified by DEAE-cellulose column. The TTP-CETPC containing fractions were eluted at 60-80 mM NaCl. The purified TTP-CETPC cysteines were oxidized to form into intermolecular disulfide bonded dimers for immunizing mice. Specific anti-CETP antibodies in mice serum were assayed by ELISA and Western blot to verify that antibodies against CETP had been successfully induced and lasted for more than seventeen weeks in vivo. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect