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 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).
Burg
M
M
Faculty of Technology, Institute of Cell Culture Technology, University of Bielefeld, Germany.
Müthing
J
J
D000818:Animals; D016466:CHO Cells; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D006224:Cricetinae; D003600:Cytosol / Q000201:enzymology; D018076:DNA, Complementary; D004587:Electrophoresis, Agar Gel; D004790:Enzyme Induction; D004926:Escherichia coli; D005982:Glutathione Transferase / Q000235:genetics; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D009439:Neuraminidase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013379:Substrate Specificity
D018076:DNA, Complementary; D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside; D005982:Glutathione Transferase; D009439:Neuraminidase