Characterization of cytosolic sialidase from Chinese hamster ovary cells Part I: Cloning and expression of soluble sialidase in Escherichia coli

The cDNA of Chinese hamster ovary (CHO) cell cytosolic sialidase was amplif ied by RT-PCR and cloned into the pGEX-2T plasmid vector encoding for gluta thione S-transferase (GST). Screening revealed transformed Escherichia coli clones with the constructed plasmid encoding the CHO cell sialidase sequen ce. After isopropyl-beta -D-thiogalactopyranoside (IPTG) induction, SDS-PAG E of the total protein extracts revealed a new protein of about 70 kDa, cor relating 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 fu sion protein was purified from sonified E. coli homogenates by one-step aff inity chromatography on Glutathione Sepharose 4B, which showed sialidase ac tivity towards 4-methyl-urnbelliferyl-alpha -D-N-acetylneuraminic acid (MUF -Neu5Ac) substrate. Induction of cells with 0.1, 0.5, and 1.0 mM IPTG revea led highest total protein amounts after induction with 1.0 mM IPTG, but hig hest specific activity for affinity chromatography purified eluates from cu ltures induced with 0.1 mM IPTG. Therefore, large scale production was perf ormed 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 degreesC. The amount of 26.46 mg of 40-fold purified GST-sialidase with a specific activ ity of 0.999 U/mg protein was obtained from crude protein extracts by one-s tep affinity chromatography. 2-Deoxy-2,3-dehydro-N-acetylneuraminic acid (N eu5Ac2en) and Neu5Ac were competitive inhibitors for the sialidase, the for mer being the more effective one using MUF-Neu5Ac as the substrate. The cyt osolic sialidase is capable of desialylating a wide spectrum of different t ypes of gangliosides using a thin-layer chromatography overlay kinetic assa y without detergents. This is the subject of the accompanying paper (Muthin g, J.; Burg, M. Carbohydr. Res. 2001., 330, 347-356). (C) 2001 Elsevier Sci ence Ltd. All rights reserved.