Thirstrup K, Carrière F, Hjorth S, Rasmussen P B, Wöldike H, Nielsen P F, Thim L
Department of Protein Chemistry, Novo Nordisk, Novo Allé, Bagsvaerd, Denmark.
FEBS Lett. 1993 Jul 19;327(1):79-84. doi: 10.1016/0014-5793(93)81044-z.
A cDNA clone encoding the sequence of human pancreatic lipase (HPL) was subcloned into the baculovirus transfer vector pVL1392 and used in co-transfection of Spodoptera frugiperda (Sf9) insect cells with wild-type Autographa californica nuclear polyhedrosis virus (AcNPV) DNA. A single recombinant protein (50 kDa) secreted by Sf9 cells was detectable in the culture medium 24 h post-infection using both anti-HPL polyclonal antibodies and potentiometric measurements of lipolytic activity. The expression level reached 40 mg/l of enzyme at 6 days. A single cation-exchange chromatography was sufficient to obtain a highly pure recombinant HPL as demonstrated by N-terminal sequencing, amino acid composition and carbohydrate analysis, as well as by mass spectrometry. These analyses revealed the production of mature protein with the correct processing of signal peptide and an homogenous glycosylation pattern. The kinetic properties of recombinant and native HPL were compared. Both enzymes showed similar profiles of interfacial activation, inhibition by bile salts and re-activation by colipase.
将编码人胰脂肪酶（HPL）序列的cDNA克隆亚克隆到杆状病毒转移载体pVL1392中，并用于与野生型苜蓿银纹夜蛾核型多角体病毒（AcNPV）DNA共转染草地贪夜蛾（Sf9）昆虫细胞。感染后24小时，使用抗HPL多克隆抗体和脂解活性的电位测量法，可在培养基中检测到Sf9细胞分泌的单一重组蛋白（50 kDa）。在第6天时，酶的表达水平达到40 mg/l。如通过N端测序、氨基酸组成和碳水化合物分析以及质谱法所证明的，单次阳离子交换色谱法足以获得高度纯化的重组HPL。这些分析揭示了成熟蛋白的产生以及信号肽的正确加工和均一的糖基化模式。比较了重组HPL和天然HPL的动力学特性。两种酶均表现出相似的界面激活、胆汁盐抑制和辅脂酶再激活特征。
