Date of Award

5-1-2004

Degree Type

Restricted Access Dissertation

Degree Name

Master of Science

Field of Study

Biomedical Sciences

Department

Graduate School of Biomedical Sciences

First Advisor

James L. Caffrey

Second Advisor

Ben G. Harris

Third Advisor

Ming-chi Wu

Abstract

Tchedre, 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.

Comments

Tchedre, 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 W 4.5 T251C 2004

Share

COinS