High-level expression and purification of a recombinant human alpha-1, 3-fucosyltransferase in baculovirus-infected insect cells.
A human alpha-1,3-fucosyltransferase (Fuc-TVII) was expressed by recombinant baculovirus-infected insect Sf9 cells as a secretory fusion protein. The fusion protein consisted of the human granulocyte colony-stimulating factor signal peptide followed by an IgG-binding domain of protein A, a Fuc-TVI-derived peptide, and the putative catalytic domain of Fuc-TVII. The signal peptide was correctly cleaved and the recombinant Fuc-TVII was secreted into the culture medium at a concentration of 10 micrograms/ml. The recombinant Fuc-TVII could be highly purified in a single-step purification procedure, i.e., IgG-Sepharose column chromatography. The enzymatic properties of the Sf9-produced Fuc-TVII were compared with the properties of that expressed by a human B-cell line, Namalwa KJM-1, transfected with an episomal plasmid carrying the fusion Fuc-TVII cDNA. Both recombinant proteins showed alpha-1,3-fucosyltransferase activity toward a type II oligosaccharide with a terminal alpha-2,3-linked sialic acid among various acceptors. The apparent Km values of Sf9-produced Fuc-TVII for GDP-fucose and its acceptor substrate were slightly lower than those of the Fuc-TVII produced by Namalwa KJM-1 cells. Sf9-produced Fuc-TVII has N-linked carbohydrate chains whose molecular weights are lower than those linked to Namalwa KJM-1-produced Fuc-TVII. This difference in carbohydrate structure hardly affects the thermal stability of Fuc-TVII. The baculovirus expression system is available for high-level expression of stable and enzymatically active secretory Fuc-TVII.
Shinkai
A
A
Tokyo Research Laboratories, Kyowa Hakko Kogyo Company, Ltd., Japan.
Shinoda
K
K
Sasaki
K
K
Morishita
Y
Y
Nishi
T
T
Matsuda
Y
Y
Takahashi
I
I
Anazawa
H
H
D000581:Amidohydrolases / Q000378:metabolism; D000818:Animals; D001402:B-Lymphocytes / Q000201:enzymology; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005647:Fucosyltransferases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D015870:Gene Expression; D006031:Glycosylation; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry; D006596:Hexosaminidases / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009439:Neuraminidase / Q000378:metabolism; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D021382:Protein Sorting Signals / Q000737:chemistry / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera / Q000235:genetics; D013205:Staphylococcal Protein A / Q000235:genetics; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured; D048809:alpha-N-Acetylgalactosaminidase
D009844:Oligosaccharides; D010446:Peptide Fragments; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D016179:Granulocyte Colony-Stimulating Factor; D005647:Fucosyltransferases; C031692:galactoside 3-fucosyltransferase; D006596:Hexosaminidases; D009439:Neuraminidase; C482722:NAGA protein, human; D048809:alpha-N-Acetylgalactosaminidase; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase