Secretion and Processing of Insulin Precursors in Yeast

A series of dibasic insulin precursors including proinsulin was expressed and secreted from Saccharomyces cerevisiae. Recombinant plasmids were constructed to encode fusion proteins consisting of a modified mating factor α 1 leader sequence and an insulin precursor. The leader sequence serves to dir...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 1986-09, Vol.83 (18), p.6766-6770
Main Authors: Thim, Lars, Hansen, Mogens T., Norris, Kjeld, Hoegh, Inge, Boel, Esper, Forstrom, John, Ammerer, Gustav, Fiil, Niels P.
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Amino acids
Biological and medical sciences
Biotechnology
Chromatography, High Pressure Liquid
Complementary DNA
Disulfides
Enzymes
Fundamental and applied biological sciences. Psychology
Genetic engineering
Genetic technics
Glucagon - biosynthesis
Humans
Insulin
Insulin - biosynthesis
Methods. Procedures. Technologies
Molecular cloning
Molecules
Phosphates
Plasmids
Proinsulin - analysis
Proinsulin - isolation & purification
Proinsulin - metabolism
Protein precursors
Saccharomyces cerevisiae - metabolism
Yeasts
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Summary:A series of dibasic insulin precursors including proinsulin was expressed and secreted from Saccharomyces cerevisiae. Recombinant plasmids were constructed to encode fusion proteins consisting of a modified mating factor α 1 leader sequence and an insulin precursor. The leader sequence serves to direct the fusion protein into the secretory pathway of the cell and to expose it to the Lys-Arg processing enzyme system. The secreted peptides were purified fromt the fermentation broth and characterized by sequencing and amino acid analysis. Processing at one or both dibasic sequences was shown in proinsulin and in other insulin precursors containing a short spacer peptide in place of the C peptide. In contrast, no processing was observed in the absence of a spacer peptide in the insulin precursor molecule, e.g., B-Lys-Arg-A (where A and B are the A and B chain of human proinsulin, respectively). This type of single-chain insulin precursors isolated from such constructions could be enzymatically converted into insulin by treatment with trypsin and carboxypeptidase B. The above results suggest that the C-peptide region of proinsulin serves to direct the trypsin-like converting enzyme to process at the two dibasic sequences. We propose that in hormone precursors in general the spacer peptides serve to expose dibasic sequences for processing.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.83.18.6766