Immunoaffinity Purification and Reconstitution of the Human Bilirubin/Phenol UDP-glucuronosyltransferase Family

• Published in: Protein expression and purification Vol. 6; no. 2; pp. 149 - 154
• Main Authors: Seppen, J., Jansen, P.L.M., Elferink, R.P.J.O.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.04.1995
• Subjects: Animals; Antibodies, Monoclonal - immunology; Bilirubin - metabolism; Chromatography, Affinity; Chromatography, Gel; Enzyme Activation; Glucuronosyltransferase - chemistry; Glucuronosyltransferase - immunology; Glucuronosyltransferase - isolation & purification; Humans; Hydrogen-Ion Concentration; Isoenzymes - chemistry; Isoenzymes - immunology; Isoenzymes - isolation & purification; Liposomes; Membrane Lipids - metabolism; Membrane Proteins - chemistry; Membrane Proteins - immunology; Membrane Proteins - isolation & purification; Mice; Mice, Inbred BALB C; Microsomes, Liver - enzymology; Octoxynol; Phenol; Phenols - metabolism; Phosphatidylcholines; Phospholipids - metabolism
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1006/prep.1995.1018

When membrane proteins are solubilized and subjected to purification procedures, the loss of lipids surrounding the protein often results in irreversible inactivation. We describe a procedure for the immunoaffinity purification of the membrane protein UDP-glucuronosyltransferase from human liver. This procedure reduces exposure of the protein to detergent, thereby reducing lipid loss. Triton X-100 was used to solubilize human Liver microsomes. The solubilized proteins were applied to a sephacryl 300 (S-300) gel filtration column equilibrated with detergent-free buffer. UDP-glucuronosyltransferase activity eluted in turbid fractions in the void volume. During passage through the column Triton X-100 can partition in the buffer, while lipids are reconstituted into vesicular structures. Proteins with a high affinity for phospholipids remain associated with the lipid and elute in the void volume. The active fractions from the S-300 column were resolubilized with Triton X-100 and applied to a column with immobilized antibody. Washing this column with buffer containing phosphatidylcholine liposomes and no detergent removed unbound proteins and minimized loss of lipid from bound UDP-glucuronosyltransferase. Raising the pH of the washing buffer to 11.5 in the presence of liposomes resulted in elution and simultaneous reconstitution of active UDP-glucuronosyltransferase. Antibodies against membrane proteins are often available but immunoaffinitypurification of active enzyme is difficult. The approach described here could be useful for the isolation of other membrane proteins.