Expression in insect cells and purification of a catalytically active recombinant human gastric lipase

Human gastric lipase (HGL) cDNA was synthesized by RT-PCR amplificationand cloned into the PVL 1392 baculovirus transfer vector. Therecombinant transfer vector was cotransfected with a modifiedbaculovirus DNA (Baculogold^TM) which contains a lethal deletion.Cotransfection of baculovirus DNA with the recombinant transfervector rescues the lethal deletion of this virus DNA and reconstitutesviable virus particles inside the transfected insect cells.BTI-TN-5B1-4 insect cells (also called High Five^TM cells) wereused to express recombinant HGL. The level of HGL secretionwas {small tilde}32 mg/1 of culture medium. The insect cellsalso accumulated HGL intracellularly, which indicated the existenceof rate-limiting steps in the secretion of HGL. Therefore weinvestigated the effect of replacing the HGL signal peptide(SP) by other SP of secreted proteins. The honeybee melittinSP and the human pancreatic lipase (HPL) SP were tested. Thefusion of HGL with HPL SP resulted in a 2-fold increase in theamount of lipase secreted from the insect cells. The recombinantactive HGL was not processed at the expected cleavage site ofthe natural enzyme, however, but at residue +3. On the otherhand, High Five^TM cells transfected with the vector encodingHGL fused to the melittin SP did not secrete any detectableactive HGL. Recombinant HGL was identified using the Westernblot procedure with rabbit polyclonal antibodies. The proteinmigrated with an apparent molecular mass of 45 kDa under SDS–PAGEanalysis (compared with 50 kDa in the case of natural HGL),indicating that the insect cells have only a limited capacityto glycosylate HGL. The maximum specific activities of the recombinantlipase were 434, 730 and 562 units/mg using long-chain (Intralipid^TM),medium-chain (trioctanoylglycerol) and short-chain (tributyroylglycerol)triacylglycerols, respectively.