Abstract
Production and purification of recombinant human lecithin:cholesterol acyltransferase (LCAT), secreted by baby hamster kidney (BHK) cells, has been improved by limiting the harvesting times for the conditioned medium and introducing an additional purification step. The recombinant BHK cells were grown until nearly confluent on multilayered flasks in a fetal-calf-serum-enriched medium. Subsequently, the cells were washed and supplied with serum free medium for 24-h periods. The conditioned medium, containing recombinant LCAT, was harvested at 24 and 48 h and subjected to chromatography on phenyl-Sepharose and ACA-44 agarose to isolate the recombinant enzyme. The second chromatography step revealed the presence of a low-molecular-weight contaminant that exhibited a carbohydrate/protein composition similar to proteoglycans. The major purified component contained LCAT activity and was homogeneous by acrylamide gel electrophoresis.
| Original language | English |
|---|---|
| Pages (from-to) | 38-41 |
| Number of pages | 4 |
| Journal | Protein Expression and Purification |
| Volume | 10 |
| Issue number | 1 |
| DOIs | |
| State | Published - 1 Jan 1997 |
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Purification of recombinant lecithin:cholesterol acyltransferase. / Nair, Maya P.; Kudchodkar, Bhalchandra J.; Pritchard, P. Haydn; Lacko, Andras G.
In: Protein Expression and Purification, Vol. 10, No. 1, 01.01.1997, p. 38-41.Research output: Contribution to journal › Article
TY - JOUR
T1 - Purification of recombinant lecithin:cholesterol acyltransferase
AU - Nair, Maya P.
AU - Kudchodkar, Bhalchandra J.
AU - Pritchard, P. Haydn
AU - Lacko, Andras G.
PY - 1997/1/1
Y1 - 1997/1/1
N2 - Production and purification of recombinant human lecithin:cholesterol acyltransferase (LCAT), secreted by baby hamster kidney (BHK) cells, has been improved by limiting the harvesting times for the conditioned medium and introducing an additional purification step. The recombinant BHK cells were grown until nearly confluent on multilayered flasks in a fetal-calf-serum-enriched medium. Subsequently, the cells were washed and supplied with serum free medium for 24-h periods. The conditioned medium, containing recombinant LCAT, was harvested at 24 and 48 h and subjected to chromatography on phenyl-Sepharose and ACA-44 agarose to isolate the recombinant enzyme. The second chromatography step revealed the presence of a low-molecular-weight contaminant that exhibited a carbohydrate/protein composition similar to proteoglycans. The major purified component contained LCAT activity and was homogeneous by acrylamide gel electrophoresis.
AB - Production and purification of recombinant human lecithin:cholesterol acyltransferase (LCAT), secreted by baby hamster kidney (BHK) cells, has been improved by limiting the harvesting times for the conditioned medium and introducing an additional purification step. The recombinant BHK cells were grown until nearly confluent on multilayered flasks in a fetal-calf-serum-enriched medium. Subsequently, the cells were washed and supplied with serum free medium for 24-h periods. The conditioned medium, containing recombinant LCAT, was harvested at 24 and 48 h and subjected to chromatography on phenyl-Sepharose and ACA-44 agarose to isolate the recombinant enzyme. The second chromatography step revealed the presence of a low-molecular-weight contaminant that exhibited a carbohydrate/protein composition similar to proteoglycans. The major purified component contained LCAT activity and was homogeneous by acrylamide gel electrophoresis.
UR - http://www.scopus.com/inward/record.url?scp=0031172307&partnerID=8YFLogxK
U2 - 10.1006/prep.1996.0711
DO - 10.1006/prep.1996.0711
M3 - Article
C2 - 9179288
AN - SCOPUS:0031172307
VL - 10
SP - 38
EP - 41
JO - Protein Expression and Purification
JF - Protein Expression and Purification
SN - 1046-5928
IS - 1
ER -