出版社：American Society for Biochemistry and Molecular Biology
摘要：We have established a baby hamster kidney (BHK) cell line that constitutively expresses significant quantities of human recombinant lecithin:cholesterol acyltransferase (rLCAT). LCAT cDNA was cloned into a mammalian expression vector containing the metallothionein promoter and the dihydrofolate reductase gene. After transfection, the BHK cells were treated with 500 microM methotrexate for 2 weeks to select the successfully transfected cells. Surviving colonies were subcloned and high level secretors were identified by measurement of LCAT activity and mass in the culture medium. The attachment of transfected cells to microcarrier beads enabled the efficient production of large quantities of rLCAT in a serum-free medium. After a single-step chromatography procedure, the rLCAT was purified to homogeneity with yields exceeding 1 mg of rLCAT per 100 ml of culture medium. The molecular weight of rLCAT (approximately 66,000) was identical to that of purified human plasma LCAT on SDS polyacrylamide electrophoresis. The rLCAT was activated by apolipoprotein A-I and had an average specific activity that was similar to purified plasma LCAT. After selective deglycosylation with either neuraminidase or N-glycanase, rLCAT and plasma LCAT had identical molecular weights. The simplification of the production and purification of rLCAT reported here will enable a more in depth analysis of the structure and function of this enzyme.
