High-Level Baculoviral Expression of Lysosomal Acid Lipase

Abstract

Lysosomal acid lipase (LAL, EC 3.1.1.13) is a hydrolase essential for the intracellular degradation of lipids derived from plasma lipoproteins (1). LAL is synthesized in most cells and tissues of the human body, including fibroblasts, macrophages and lymphocytes. Although LAL activity is easily detected in these tissues, detailed physiological studies have been hindered by low levels of tissue expression. Moreover, biochemical purification strategies have suffered from the instability of the lipase such that structural and functional features of LAL have not been investigated to any great extent. Short-time expression systems generating recombinant enzyme in encaryotic cells, while suitable for studying effects of mutations of enzyme activities (2), are limited by their low efficiency of enzyme production precluding purification and biochemical analysis of LAL. Attempts to express LAL in bacteria failed due to precipitation of heterologous protein in inclusion bodies. In addition, bacterial expression systems may not be suitable to produce enzymatically active LAL since bacterial cells do not provide co- and posttranslational modifications necessary for enzyme activity. In contrast, transient protein expression in insect cells mediated by baculoviral vectors is characterized by high levels of recombinant protein production with suitable posttranslational modification (3), allowing purification of expressed protein from cell extracts or culture supernatants.