Secretion of active human lecithin-cholesterol acyltransferase by insect cells infected with a recombinant baculovirus

Abstract

Difficulties in purifying the plasma enzyme lecithin-cholesterol acyltransferase (LCAT) have hampered detailed studies of its (patho)physiological role in lipoprotein metabolism and of structure-function relationships. Potentially, baculovirus-driven expression systems offer a powerful means to produce significant amounts of LCAT. Accordingly, full-length LCAT cDNA was cloned into pVL 1392, a high-level expression derivative of Autographa californica nuclear polyhedrosis virus (AcNPV), and the resultant plasmid was co-transfected into Trichoplusia ni insect cells (High 5 line) with a linearized viral DNA using lipofectin. Such viral DNA had a lethal mutation and grew only when recombined with a pVL1392-type rescue plasmid; cells infected with recombinant Autographa californica LCAT virus changed from a fibroblast-like morphology to rounded, but lacked the polyhedrin occlusion bodies characteristic of wild-type AcNPV infections. Enzymically active recombinant LCAT (rLCAT), sensitive to sulphydryl reagents, was secreted in the late phase of infection (36-48 h) but was absent with wild-type infections. The secreted protein had an apparent molecular mass of 53 kDa by SDS/PAGE, lower than that of native plasma LCAT; it was susceptible to endoglycosidase H digestion and was bound by concanavalin A, suggesting that precursor high-mannose N-glycan chains had not undergone full maturation to complex types. Pretreatment of the cells with tunicamycin to inhibit the first step of N-glycosylation led to intracellular accumulation of immature rLCAT (approximately 46-48 kDa) and a marked reduction in enzyme secreted. We conclude that the baculovirus gene-expression system will permit production of biologically active normal and mutant forms of LCAT protein.

Production and characterization of the Brassica oleracea self-incompatibility locus glycoprotein and receptor kinase in a baculovirus infected insect cell culture system

“…3c, lanes 15 and 17). The observation that the bulk of the undegraded recombinant protein produced in insect cells is unglycosylated, and possibly misfolded, is not unusual, and has been reported elsewhere (Chawla and Owen 1995;Tate and Blakely 1994). This effect may reflect an overloading of the glycosylation or 1,2,7,8,13,14), pAc-SRK 6 virus (lanes 3-6), the pAc-SLG 2 virus (lanes 9-12), or the pAc-SLG 6 virus (lanes 15-18) secretion machinery by the large amounts of heterologous proteins produced (Jarvis et al 1990).…”

C-Terminal His6-Tagged Lecithin-Cholesterol Acyltransferase (LCAT) is Catalytically Active

“…Secreted LCAT protein was estimated by comparison of its enzymic activity with human plasma (6 pg/ml), assuming the Same specific activity [7].…”

“…Recently, we expressed recombinant LCAT in insect cells [7] and in mammalian (Chinese hamster ovary, CHO) cells. However, despite high production of LCAT by these methods (10-20 mg/litre), isolation of the enzyme has still proved problematical; to ensure pure LCAT, the final step has required CHT-I1 hydroxyapatite chromatography (BioRad) which results in a low recovery.…”

Effects of peroxynitrite on plasma components of the reverse cholesterol transport pathway

“…Differential ultracentrifugation flotation was used to isolate HDL 3 (1.125–1.26 g/ml) from freshly isolated plasma [37]. Recombinant human LCAT was isolated from the serum‐free culture medium of Chinese hamster ovary (CHO) cells [38], by phenyl‐Sepharose chromatography [39, 40].…”