Conversion of a bovine UDP-GalNAc:polypeptide, N-acetylgalactosaminyltransferase to a soluble, secreted enzyme, and expression in Sf9 cells.
A soluble, secreted UDP-GalNAc:polypeptide, N-acetylgalactosaminyltransferase, was prepared by substituting the honeybee melittin leader sequence for the sequences coding for the cytoplasmic and membrane spanning domains of a cloned, bovine full-length cDNA (F. L. Homa et al., 1993, J. Biol. Chem. 268, 12609-12616). When this construct was expressed in insect cells using a recombinant baculovirus, a fully active soluble enzyme was recovered from the culture medium. In large-scale preparations, approximately 2-3 mg of enzyme protein was produced per liter of medium. A one-step purification of the soluble molecule on apomucin-Sepharose yielded an essentially homogeneous enzyme preparation. The purified soluble enzyme has a molecular mass of approximately 61 kDa and appears to contain both N- an O-linked oligosaccharides. NH2-terminal sequencing demonstrated that the melittin leader sequence is cleaved at the predicted site and amino acid analysis gave results in close agreement with the composition predicted by the nucleic acid sequence. The enzymatic properties of the soluble, recombinant molecule are similar to those of the enzymes isolated from bovine colostrum and porcine submaxillary gland: the specific activity is approximately 2160 U/mg protein and the Kms for UDP-GalNAc and the synthetic acceptor peptides PPASTSAPG and PPDAASAAPLR are approximately 1.7 microM, 6.5 mM, and 3.6 mM, respectively.
Homa
F L
FL
Upjohn Company, Upjohn Laboratories, Kalamazoo, Michigan 49001, USA.
Baker
C A
CA
Thomsen
D R
DR
Elhammer
A P
AP
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle / Q000235:genetics*; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D005813:Genes, Synthetic; D005822:Genetic Vectors; D006031:Glycosylation; D008555:Melitten / Q000235:genetics; D008969:Molecular Sequence Data; D017350:N-Acetylgalactosaminyltransferases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017924:Nucleopolyhedroviruses; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D012995:Solubility; D018411:Spodoptera; D000097763:Polypeptide N-acetylgalactosaminyltransferase
D018076:DNA, Complementary; D008555:Melitten; D017350:N-Acetylgalactosaminyltransferases; D011993:Recombinant Fusion Proteins; D000097763:Polypeptide N-acetylgalactosaminyltransferase