Browsing by Subject "cholesterol acyltransferase"
Now showing 1 - 2 of 2
- Results Per Page
- Sort Options
Item Characterization of Recombinant Lecithin: Cholesterol Acyltransferase, Secreted by a Human Lung Cell Line (1069-111) and by Pichia Pastoris Yeast Cells(2004-05-01) Tchedre, Kissaou T.; Caffrey, James L.; Harris, Ben G.; Wu, Ming-ChiTchedre, Kissaou T., Characterization of Recombinant Lecithin: Cholesterol Acyltransferase, Secreted by a Human Lung Cell Line (1069-111) and by Pichia pastoris Yeast Cells (Biomedical Sciences), May, 2004, Lecithin: cholesterol acyltransferase (LCAT) is a key enzyme in mammalian lipoprotein metabolism. Associated with the surface of high-density lipoproteins (HDL), LCAT contributes to the homeostasis of circulating free and esterified cholesterol via the reverse cholesterol transport pathway. The purpose of these studies was to characterize a recombinant form of LCAT, secreted by a human lung cell line (Beta gene 1069/111) and to evaluate a new expression system for LCAT using transformed Pichia pastoris cells. A human lung cell line (Beta gene 1069/111), transfected with pBIISK (Stratagene)+ vector was used as the source of recombinant (rLCAT) for the first stage of characterization studies. Human lung cells were expanded in Dulbecco’s minimal essential medium (DMEM) supplemented with 10% fetal bovine serum for the expression of the recombinant LCAT. At 80 – 90% confluency, the medium was changed to a serum free preparation and the flasks were incubated for 48 hrs at 37°C to facilitate the secretion of the enzyme. Beta gene (1069/111) LCAT was purified from the conditioned medium using phenyl sepharose chromatography. The purified enzyme was characterized according to: carbohydrate composition, and enzyme kinetic parameters. The enzymatic characteristics, of the human lung cell line LCAT had similar Km and Vmax values to other LCAT preparations, isolated from other expression systems and human plasma. Deglycosylation reduced the molecular weight of the enzyme from about 67,000 to about 43,000 suggesting a carbohydrate component of 25-32% of the enzyme’s total mass. Detailed analysis of the carbohydrate structures revealed N-glycan structures in a complex pattern of sialylated and fucosylated tri and tetra-antennary glycosides (8). In addition to the Beta gene expression, a Pichia pastoris yeast expression system was also developed consisting of human LCAT cDNA cloned into pPICZαA vector along with a removable amino-terminal polyhistidine tag. The Pichia pastoris cells were transformed with a vector containing the LCAT gene cDNA and transformants were selected on agar plates containing zeocine (100μg/ml). Polymerase chain reaction (PCR) and reverse transcription polymerase chain reaction (RT-PCR) were used to confirm the correct integration of the LCAT gene cDNA into the pPICZαA vector. The recombinant LCAT produced by the yeast cultures was purified by Talon affinity chromatography, taking advantage of the removable histidine tag. The enzymatic activity was determined using proteoliposome vesicles. The Yeast expression system yielded ~18 mg of enzyme protein/500 ml and thus may provide an appropriate enzyme source for characterization studies via NMR analysis and x-ray crystallography.Item Horse Serum High Density Lipoproteins as Drug Transporters(2004-05-01) Johnson, Shemedia; Walter McConathyJohnson, Shemedia J., Horse Serum High Density Lipoproteins (HDL) as Drug Transporters. High-density lipoproteins (HDL) are complex particles composed of specific proteins and lipids that facilitate blood and tissue cholesterol homeostasis by transporting excess peripheral cholesterol to the liver. In association with cholesterol ester transfer protein (CETP) and the enzyme, lecithin: cholesterol acyltransferase (LCAT), HDL contributes to the transport of hydrophobic lipids, including cholesterol ester and triglycerides through the blood. The studies presented here involve the evaluation of horse serum HDL as a carrier of water insoluble drugs and an improved process to isolate and purify horse serum HDL utilizing hydrophobic affinity chromatography. Dilauryl fluorescein (DLF) has been chosen as a model compound for the study of horse HDL as a drug carrier. The prepared HDL/DLF particles have similar flotation densities and size properties to native horse serum HDL. The amount of DLF incorporated into HDL is 30μg/mg protein. Various cancer cell lines internalized DLF from horse HDL/DLF particles successfully. While human plasma contains cholesterol ester transfer protein (CETP), horse plasma does not. Horse plasma/serum can be supplemented by human plasma to study the role of CETP in drug transport and the stability of the horse HDL/drug complex.