Expressing and purifying an anti-atherosclerosis polypeptide vaccine in Escherichia coli

Protein Expression and Purification
Qi GaofuLiu Jingjing

Abstract

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 m...Continue Reading

References

Sep 1, 1994·Clinical and Experimental Pharmacology & Physiology·P Barter, K A Rye
Sep 9, 2000·Arteriosclerosis, Thrombosis, and Vascular Biology·C W RittershausU S Ryan
Apr 26, 2003·Atherosclerosis. Supplements·Bela F Asztalos, Ernst J Schaefer

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Citations

Jan 25, 2013·Immunologic Research·Chrysoulla PieridesErnesto Oviedo-Orta
Nov 8, 2016·Expert Review of Vaccines·Dania O Govea-AlonsoSergio Rosales-Mendoza
Aug 11, 2006·Current Opinion in Cardiology·Enrique P Gurfinkel, Veronica S Lernoud
Sep 18, 2007·American Journal of Physiology. Heart and Circulatory Physiology·Carlos BecharaChangyi Chen

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