TY - JOUR
T1 - Immunoaffinity purification and reconstitution of the human bilirubin/phenol UDP-glucuronosyltransferase family
AU - Seppen, J.
AU - Jansen, P. L.
AU - Oude Elferink, R. P.
N1 - Copyright: Copyright 2017 Elsevier B.V., All rights reserved.
PY - 1995/4
Y1 - 1995/4
N2 - 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
AB - 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
UR - http://www.scopus.com/inward/record.url?scp=0029278867&partnerID=8YFLogxK
U2 - https://doi.org/10.1006/prep.1995.1018
DO - https://doi.org/10.1006/prep.1995.1018
M3 - Article
C2 - 7606162
SN - 1046-5928
VL - 6
SP - 149
EP - 154
JO - Protein Expression and Purification
JF - Protein Expression and Purification
IS - 2
ER -