[13] Hepatic lipase: High-level expression and subunit structure determination

This chapter describes procedures developed for a large-scale production and purification of recombinant hepatic lipase (HL). The purification of sufficient quantities of this enzyme has enabled the analysis of its subunit structure by three independent means: intensity light scattering, sedimentation equilibrium, and radiation inactivation. The low protein yields associated with the purification of HL from human postheparin plasma have not permitted a more detailed physical analysis of this enzyme. For example, the only previous estimations of the functional molecular weight of HL have relied on gel-filtration techniques. The primary disadvantage of conventional size-exclusion chromatography methods is that the elution position depends not only on the molecular weight of the molecule, but also on its shape. In addition, if the protein has a tendency to adhere to the column matrix, the molecular weight may be underestimated.