Cloning and expression of a marine bacterial beta-galactoside alpha2,6-sialyltransferase gene from Photobacterium damsela JT0160.
Sialyltransferase 0160, a bacterial sialyltransferase which catalyzes the incorporation of NeuAc from CMP-NeuAc into the galactose residue of the carbohydrate chain at position 6, is produced by Photobacterium damsela JT0160. The gene coding for sialyltransferase 0160 (bst) was cloned, sequenced, and expressed in Escherichia coli. The sialyltransferase 0160 gene contains an open reading frame of 2,028 base pairs encoding a protein of 675 amino acid residues. The deduced amino acid sequence of sialyltransferase 0160 did not contain the sialylmotif and had no significant similarity to mammalian sialyltransferases. Crude extracts of cultured E. coli MV1184 cells carrying an expression plasmid for the sialyltransferase 0160 gene showed sialyltransferase activity, which was identified as beta-galactoside alpha2,6-sialyltransferase activity by enzymatic reaction product analysis. In addition, when mutant genes, lacking 3'-coding regions for COOH-terminal portions of the protein, which are thought to form alpha-helix structures, were expressed in E. coli MV1184, soluble-form enzymes were obtained. This implies that the COOH-terminal portion of sialyltransferase 0160 is required for membrane binding.
Yamamoto
T
T
Sea Water Science Research Laboratory, Japan Tobacco Inc., Odawara, Kanagawa.
Nakashizuka
M
M
Terada
I
I
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004926:Escherichia coli; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010776:Photobacterium / Q000201:enzymology* / Q000235:genetics; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D012799:Sialyltransferases / Q000235:genetics* / Q000302:isolation & purification; D012995:Solubility; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D012799:Sialyltransferases; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase