PURIFICATION AND INTERFACIAL BEHAVIOR OF RECOMBINANT HUMAN GASTRIC LIPASE PRODUCED FROM INSECT CELLS IN A BIOREACTOR

Recombinant human gastric lipase (rHGL) (EC 3.1.1.3) was produced on a large scale (5-13 mg/liter) from recombinant baculovirus-infected ins ect cells using a bioreactor apparatus. Here an improved procedure is described for purifying rHGL involving the use of cation exchange chro matography followed by immunoaffinity column methods, which gives a to tal yield of 62% and a purification factor of 464, using 10% isopropan ol in all the purification buffers. The presence of isopropanol was ne cessary to preserve the stability of the enzyme during the chromatogra phic separation steps. The specific activity of rHGL on tributyroylgly cerol (700 U/mg) was lower than that of native HGL (nHGL) (1080 U/mg), The rHGL interfacial adsorption kinetics were studied by recording th e changes in the surface pressure with time in the presence or absence of an egg phosphatidycholine monomolecular film spread at the air/wat er interface at various initial surface pressures. The surface behavio r of rHGL was similar to that of nHGL. It can be concluded that the li pid binding affinity of rHGL is identical to that of the native lipase and, consequently, that the presence of detergents and lipids in the insect cell culture media did not affect the interfacial behavior of t he purified rHGL. It will be therefore possible to specifically study the binding step of HGL mutants to a lipid monolayer. (C) 1998 Academi c Press.