Wicker-Planquart C, Canaan S, Rivière M, Dupuis L, Verger R
Laboratoire de Lipolyse Enzymatique, UPR 9025, de l'IFR-1 du CNRS, Marseille, France.
Protein Eng. 1996 Dec;9(12):1225-32. doi: 10.1093/protein/9.12.1225.
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.
通过逆转录聚合酶链反应（RT-PCR）扩增合成人胃脂肪酶（HGL）cDNA，并将其克隆到PVL 1392杆状病毒转移载体中。将重组转移载体与含有致死性缺失的修饰杆状病毒DNA（Baculogold）共转染。杆状病毒DNA与重组转移载体的共转染挽救了该病毒DNA的致死性缺失，并在转染的昆虫细胞内重新构建了有活力的病毒颗粒。使用BTI-TN-5B1-4昆虫细胞（也称为High Five细胞）来表达重组HGL。HGL的分泌水平约为每升培养基32毫克。昆虫细胞还在细胞内积累HGL，这表明HGL分泌过程中存在限速步骤。因此，我们研究了用其他分泌蛋白的信号肽（SP）替换HGL信号肽的效果。测试了蜜蜂蜂毒肽SP和人胰脂肪酶（HPL）SP。HGL与HPL SP的融合导致昆虫细胞分泌的脂肪酶量增加了2倍。然而，重组活性HGL并非在天然酶预期的切割位点进行加工，而是在+3位残基处。另一方面，用编码与蜂毒肽SP融合的HGL的载体转染的High Five细胞未分泌任何可检测到的活性HGL。使用兔多克隆抗体通过蛋白质印迹法鉴定重组HGL。在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析下，该蛋白迁移时的表观分子量为45 kDa（天然HGL为50 kDa），表明昆虫细胞对HGL进行糖基化的能力有限。使用长链（英脱利匹特）、中链（三辛酰甘油）和短链（三丁酰甘油）三酰甘油时，重组脂肪酶的最大比活性分别为434、730和562单位/毫克。
