Alpha-factor leader sequence-directed transport of Escherichia coli beta-galactosidase in the secretory pathway of Saccharomyces cerevisiae.
The construction of two fused genes is described. One involves the in-frame fusion of the yeast prepro-alpha-factor coding sequence, and the Escherichia coli lac Z gene. The second gene fusion utilizes a 103 bp yeast invertase NH2-terminal coding sequence at the fusion junction of the hybrid gene described above. The gene fusions, under the control of the alpha-factor promoter, expressed active beta-galactosidase in alpha haploid yeast cells. The activity could be regulated in a temperature-sensitive sir3 mutant. The incorporation of the invertase coding sequence at the MF alpha 1-lacZ fusion junction provided significantly higher levels of beta-galactosidase activity. A substantial quantity of the hybrid proteins generated from the gene fusions was primarily localized in the intracellular membranes of yeast cells, while a processed form could be secreted into the periplasm.
Das
R C
RC
Research and Development, Miles Inc., Elkhart, IN 46515.
Shultz
J L
JL
Lehman
D J
DJ
D015152:Blotting, Northern; D015153:Blotting, Western; D002458:Cell Fractionation; D003001:Cloning, Molecular; D003470:Culture Media; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005696:Galactosidases / Q000235:genetics*; D005786:Gene Expression Regulation; D000072235:Mating Factor; D010455:Peptides / Q000235:genetics*; D011233:Precipitin Tests; D011993:Recombinant Fusion Proteins / Q000032:analysis; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013347:Subcellular Fractions / Q000032:analysis; D001616:beta-Galactosidase / Q000235:genetics*
D003470:Culture Media; D010455:Peptides; D011993:Recombinant Fusion Proteins; D000072235:Mating Factor; D005696:Galactosidases; D001616:beta-Galactosidase