Expression in insect cells and purification of a catalytically active recombinant human gastric lipase.
Human gastric lipase (HGL) cDNA was synthesized by RT-PCR amplification and cloned into the PVL 1392 baculovirus transfer vector. The recombinant transfer vector was cotransfected with a modified baculovirus DNA (Baculogold) which contains a lethal deletion. Cotransfection of baculovirus DNA with the recombinant transfer vector rescues the lethal deletion of this virus DNA and reconstitutes viable virus particles inside the transfected insect cells. BTI-TN-5B1-4 insect cells (also called High Five cells) were used to express recombinant HGL. The level of HGL secretion was approximately 32 mg/l of culture medium. The insect cells also accumulated HGL intracellularly, which indicated the existence of rate-limiting steps in the secretion of HGL. Therefore we investigated the effect of replacing the HGL signal peptide (SP) by other SP of secreted proteins. The honeybee melittin SP and the human pancreatic lipase (HPL) SP were tested. The fusion of HGL with HPL SP resulted in a 2-fold increase in the amount of lipase secreted from the insect cells. The recombinant active HGL was not processed at the expected cleavage site of the natural enzyme, however, but at residue +3. On the other hand, High Five cells transfected with the vector encoding HGL fused to the melittin SP did not secrete any detectable active HGL. Recombinant HGL was identified using the Western blot procedure with rabbit polyclonal antibodies. The protein migrated with an apparent molecular mass of 45 kDa under SDS-PAGE analysis (compared with 50 kDa in the case of natural HGL), indicating that the insect cells have only a limited capacity to glycosylate HGL. The maximum specific activities of the recombinant lipase were 434, 730 and 562 units/mg using long-chain (Intralipid), medium-chain (trioctanoylglycerol) and short-chain (tributyroylglycerol) triacylglycerols, respectively.
Wicker-Planquart
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Laboratoire de Lipolyse Enzymatique, UPR 9025, de l'IFR-1 du CNRS, Marseille, France.
Canaan
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Rivière
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Dupuis
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Verger
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D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D005822:Genetic Vectors; D006031:Glycosylation; D006801:Humans; D008049:Lipase / Q000096:biosynthesis* / Q000235:genetics; D050356:Lipid Metabolism; D008555:Melitten / Q000235:genetics; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000096:biosynthesis; D018411:Spodoptera / Q000166:cytology / Q000821:virology; D013270:Stomach / Q000201:enzymology*; D013379:Substrate Specificity
D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D008555:Melitten; D008049:Lipase