Expression and secretion of heterologous proteins in yeast employing truncated alpha-factor leader sequences

a technology of alpha-factor leader sequence and heterologous protein, which is applied in the field of recombinant proteins production in yeast, can solve the problems of not having, not generally predictable, heterologous protein in the patent, and achieve the effect of efficient direct expression and secretion of heterologous polypeptides, and improving the efficiency of expression

Inactive Publication Date: 2001-08-28
CHIRON CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

It has been surprisingly discovered that a truncated form of the .alpha.-factor leader sequence can efficiently direct the expression and secretion of heterologous polypeptides in yeast. Particularly surprising is the discovery that truncated .alpha.-factor leader sequences can substantially improve the efficiency of expression of such heterologous proteins in relation to expression systems using t

Problems solved by technology

The patent, however, does not contain data which would indicate that the patentees ever successfully employed the .alpha.-factor leader to express and secrete a heterologous protein in yeast.
While the above work demonstrates that the .alpha.-factor expression system is widely useful, it is not generally predicta

Method used

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  • Expression and secretion of heterologous proteins in yeast employing truncated alpha-factor leader sequences
  • Expression and secretion of heterologous proteins in yeast employing truncated alpha-factor leader sequences
  • Expression and secretion of heterologous proteins in yeast employing truncated alpha-factor leader sequences

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examples

The following examples are provided for illustrative purposes only, and are not intended to limit the scope of the present invention. It is believed that the deposit of the starting biological materials is not necessary for the practice of the present invention since either the same or equivalent materials are publicly available.

example i

The following example provides a comparison of the levels of expression and secretion obtained with modified .alpha.-factor constructs used to express human proinsulin. Three constructs employ full-length .alpha.-factor leaders; one having .alpha.-factor leader with the three native glycosylation sites, one having all three of the glycosylation sites eliminated, and one having all of the sites, except the one at Asn.sub.23, removed. The fourth construct is a truncated .alpha.-factor leader which retains a single glycosylation site at Asn.sub.23.

A. pYGAI1

This plasmid encodes an .alpha.-factor leader [Brake et al. (1984) Proc. Natl. Acad. Sci. USA 81:4642-4646; EPO Publication No. 116,201] linked to human proinsulin. The proinsulin is encoded by a synthetic gene made with yeast preferred codons (FIG. 1). The .alpha.-factor leader sequence, the synthetic proinsulin gene and the .alpha.-factor terminator sequence are from pYBCA5, the construction of which is shown in FIG. 1. Transcripti...

example ii

This example compares the expression of a full-length .alpha.-factor leader construct, retaining all glycosylation sites, to an expression construct employing a truncated .alpha.-factor sequence retaining only a single glycosylation site at Asn.sub.23. The non-yeast protein employed in this example is a human proinsulin analog wherein the connecting "C" peptide has been replaced by a yeast KEX2 endopeptidase cleavage site.

A. pYGAIC3

The plasmid pGAIC3 was made by replacing the 231 bp HindIII-SalI fragment of pGAI1 which encodes amino acids 14 through 30 of the B chain, the C-peptide, the A chain and 2 translation stop codons with a 132 bp synthetic HindIII-SalI gene fragment (shown in FIG. 3) which encodes amino acids 14 through 30 of the B chain, a Lys-Arg KEX2 endopeptidase cleavage site, the A chain, and translation stop codons. The plasmid pYGAIC3 was prepared from pGAIC3 as follows.

Plasmid pGAIC3 was digested with BamHI, and the 1107 bp BamHI expression cassette containing the G...

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Abstract

A yeast alpha-factor expression system is provided comprised of a truncated leader sequence, containing the alpha-factor signal peptide and one glycosylation site, linked by a processing site to a non-yeast protein-encoding sequence.

Description

Related U.S. Application DataThis is a reissue of U.S. Pat. No. 5,602,034, Issued, Feb. 11, 1997, incorporated herein by reference in its entirety.TECHNICAL FIELDThe present invention relates to the production of recombinant proteins in yeast. More particularly, the present invention is directed to an improved .alpha.-factor expression system which provides for the secretion of heterologous proteins from yeast host cells.BACKGROUNDKurjan et al. (1982) Cell 30:933-943 discloses the first cloning and sequencing of a gene encoding a yeast .alpha.-factor precursor gene. Kurjan et al., U.S. Pat. No. 4,546,082, also reports the cloning of this gene, and suggests that the .alpha.-factor leader sequence can be employed to direct the secretion of heterologous proteins in yeast. The patent, however, does not contain data which would indicate that the patentees ever successfully employed the .alpha.-factor leader to express and secrete a heterologous protein in yeast.EPO Publication No. 116,20...

Claims

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Application Information

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IPC IPC(8): C07K14/435C07K14/39C07K14/37C07K14/62C07K14/65C12N15/81C12N1/15C12N15/09C12N1/19C12P21/00C12P21/02C12R1/865
CPCC07K14/39C07K14/62C07K14/65C07K2319/02C12N15/81
Inventor TEKAMP-OLSON, PATRICIA
Owner CHIRON CORP
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