中国仓鼠卵巢细胞胞质唾液酸酶的特性：第一部分：可溶性唾液酸酶在大肠杆菌中的克隆与表达

Faculty of Technology, Institute of Cell Culture Technology, University of Bielefeld, Germany.

Carbohydr Res. 2001 Feb 15;330(3):335-46. doi: 10.1016/s0008-6215(00)00294-9.

The cDNA of Chinese hamster ovary (CHO) cell cytosolic sialidase was amplified by RT-PCR and cloned into the pGEX-2T plasmid vector encoding for glutathione S-transferase (GST). Screening revealed transformed Escherichia coli clones with the constructed plasmid encoding the CHO cell sialidase sequence. After isopropyl-beta-D-thiogalactopyranoside (IPTG) induction, SDS-PAGE of the total protein extracts revealed a new protein of about 70 kDa, correlating with the molecular weight of a fusion protein composed of the GST (26 kDa) and the cloned cytosolic CHO cell sialidase (43 kDa). A soluble fusion protein was purified from sonified E. coli homogenates by one-step affinity chromatography on Glutathione Sepharose 4B, which showed sialidase activity towards 4-methyl-umbelliferyl-alpha-D-N-acetylneuraminic acid (MUF-Neu5Ac) substrate. Induction of cells with 0.1, 0.5, and 1.0 mM IPTG revealed highest total protein amounts after induction with 1.0 mM IPTG, but highest specific activity for affinity chromatography purified eluates from cultures induced with 0.1 mM IPTG. Therefore, large scale production was performed by inducing cells during exponential growth in a 25 L bioreactor for 3 h with 0.1 mM IPTG after chilling the cell suspension to 25 degrees C. The amount of 26.46 mg of 40-fold purified GST-sialidase with a specific activity of 0.999 U/mg protein was obtained from crude protein extracts by one-step affinity chromatography. 2-Deoxy-2,3-dehydro-N-acetylneuraminic acid (Neu5Ac2en) and Neu5Ac were competitive inhibitors for the sialidase, the former being the more effective one using MUF-Neu5Ac as the substrate. The cytosolic sialidase is capable of desialylating a wide spectrum of different types of gangliosides using a thin-layer chromatography overlay kinetic assay without detergents. This is the subject of the accompanying paper (Müthing, J.; Burg, M. Carbohydr. Res. 2001, 330, 347-356).

通过逆转录聚合酶链反应（RT-PCR）扩增中国仓鼠卵巢（CHO）细胞胞质唾液酸酶的互补脱氧核糖核酸（cDNA），并将其克隆到编码谷胱甘肽S-转移酶（GST）的pGEX-2T质粒载体中。筛选显示转化的大肠杆菌克隆带有构建的编码CHO细胞唾液酸酶序列的质粒。用异丙基-β-D-硫代半乳糖苷（IPTG）诱导后，总蛋白提取物的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）显示出一种约70 kDa的新蛋白，这与由GST（26 kDa）和克隆的CHO细胞胞质唾液酸酶（43 kDa）组成的融合蛋白的分子量相关。通过在谷胱甘肽琼脂糖4B上进行一步亲和层析，从超声破碎的大肠杆菌匀浆中纯化出一种可溶性融合蛋白，该蛋白对4-甲基伞形酮基-α-D-N-乙酰神经氨酸（MUF-Neu5Ac）底物表现出唾液酸酶活性。用0.1、0.5和1.0 mM IPTG诱导细胞，结果显示用1.0 mM IPTG诱导后总蛋白量最高，但从用0.1 mM IPTG诱导的培养物中亲和层析纯化的洗脱液的比活性最高。因此，在将细胞悬液冷却至25℃后，在25 L生物反应器中于指数生长期用0.1 mM IPTG诱导细胞3小时进行大规模生产。通过一步亲和层析从粗蛋白提取物中获得了26.46 mg 40倍纯化的GST-唾液酸酶，其比活性为0.999 U/mg蛋白。2-脱氧-2,3-脱氢-N-乙酰神经氨酸（Neu5Ac2en）和Neu5Ac是唾液酸酶的竞争性抑制剂，以前者作为MUF-Neu5Ac底物时更有效。使用无去污剂的薄层色谱覆盖动力学测定法，胞质唾液酸酶能够使多种不同类型的神经节苷脂去唾液酸化。这是随附论文（Müthing, J.; Burg, M. Carbohydr. Res. 2001, 330, 347 - 356）的主题。