Addition of a dipeptide spacer significantly improves secretion of ovine trophoblast interferon in yeast.
Yeast has been analysed for its potential to secrete an ovine member of the type-I interferon (IFN) family, trophoblastin (oTP-1). The processing potential of the yeast KEX2 gene product (KEX2p) was evaluated using gene oTP-1 fused to the pre-pro sequence encoding the pre-pro peptide of the yeast alpha-factor precursor. High-level accumulation of nonprocessed (unmatured) recombinant oTP-1 (re-oTP-1) was observed in the medium. In order to short-circuit the limiting activity of KEX2p and to obtain a fully matured re-oTP-1, secretion was directed using a pre::oTP-1 fusion, relying only on signal peptidase-dependent processing. However, secretion of oTP-1 was impaired. High-level secretion was restored when the gene product contained a peptide spacer between oTP-1 and the signal peptidase cleavage site. The oTP-1 variant was shown to have an extended N terminus. An N-extended form was examined further and shown to have the correct size. Surprisingly, the variant retained its in vitro and in vivo biological activities. This system is likely to represent a general method for high-level secretion of type-I IFNs.
Degryse
E
E
Department of Molecular Biology, Transgene S.A., Strasbourg, France.
Dietrich
M
M
Nguyen
M
M
Achstetter
T
T
Charlier
M
M
Charpigny
G
G
Gaye
P
P
Martal
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004274:DNA, Recombinant / Q000235:genetics; D004151:Dipeptides / Q000235:genetics; D010450:Endopeptidases / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D007370:Interferon Type I / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D011257:Pregnancy Proteins / Q000235:genetics / Q000378:metabolism*; D011498:Protein Precursors / Q000096:biosynthesis / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases; D012756:Sheep / Q000235:genetics*; D014170:Transformation, Genetic; D015003:Yeasts / Q000235:genetics
D004274:DNA, Recombinant; D004151:Dipeptides; D007370:Interferon Type I; D008565:Membrane Proteins; D011257:Pregnancy Proteins; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C020947:interferon tau; D010450:Endopeptidases; D012697:Serine Endopeptidases; C026515:type I signal peptidase