Expressing and purifying an anti-atherosclerosis polypeptide vaccine in Escherichia coli.
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.
Gaofu
Qi
Q
Minigene Pharmacy Laboratory, Biopharmaceutical College, China Pharmaceutical University, Nanjing, PR China. garyq_2001@yahoo.com
Jie
Wu
W
Xin
Yang
Y
Roque
Rouel S
RS
Jingjing
Liu
L
D000595:Amino Acid Sequence; D000818:Animals; D000917:Antibody Formation / Q000276:immunology; D001161:Arteriosclerosis / Q000276:immunology*; D001224:Aspartic Acid / Q000737:chemistry; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D002352:Carrier Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D053480:Cholesterol Ester Transfer Proteins; D019281:Dimerization; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D006023:Glycoproteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D006851:Hydrochloric Acid / Q000737:chemistry; D007114:Immunization; D002479:Inclusion Bodies / Q000737:chemistry / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D011392:Proline / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D013744:Tetanus Toxin / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D022223:Vaccines, Subunit / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
D001426:Bacterial Proteins; C506642:CETP protein, human; D002352:Carrier Proteins; D053480:Cholesterol Ester Transfer Proteins; D006023:Glycoproteins; D011993:Recombinant Fusion Proteins; D013744:Tetanus Toxin; D022223:Vaccines, Subunit; D001224:Aspartic Acid; D011392:Proline; D006851:Hydrochloric Acid