Changes in a mammalian signal sequence required for efficient protein secretion by yeasts.
A plasmid-encoded gene for a hybrid pre-protein containing most of the bovine prolactin signal peptide (SpPRL) fused to the mature sequence of yeast invertase (IVT) was expressed and the product was processed and secreted by yeast. However, the level of IVT activity was reduced about six-fold when compared to that obtained with the wild type (wt) invertase signal peptide (SpIVT). When the 5'-untranslated sequence of the hybrid mRNA was truncated by 29 nucleotides, a 2.5-fold increase in secreted IVT was observed. Replacement of the PRL codons with preferred yeast codons did not result in any improvement in the production of secreted IVT. An increase in IVT activity to the level observed with the wt SpIVT was obtained by replacement of the Gly residue located between the N terminus and the central lipophilic region of the SpPRL by Ala. Since this amino acid replacement results in a higher probability of the SpPRL assuming an alpha-helical conformation, it suggests that the secondary structure of this region is important in recognition by the yeast secretory apparatus.
Ngsee
J K
JK
Department of Biological Sciences, Stanford University, CA 94305-5020.
Smith
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular / Q000379:methods*; D003062:Codon / Q000235:genetics; D006026:Glycoside Hydrolases / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D011388:Prolactin / Q000096:biosynthesis / Q000235:genetics*; D011487:Protein Conformation; D021382:Protein Sorting Signals / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D043324:beta-Fructofuranosidase
D003062:Codon; D021382:Protein Sorting Signals; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D011388:Prolactin; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase