Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-03-25
2001-03-06
McKelvey, Terry (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S254200, C435S254210, C435S254220, C435S254230, C435S471000
Reexamination Certificate
active
06197545
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention provides a genetically engineered yeast cell comprising a eukaryotic homologue of a yeast signal peptidase complex subunit. Specifically, this invention provides a yeast cell having functional subunits of the yeast signal peptidase complex, comprising eukaryotic homologues of the yeast Sec11p, Spc1p, Spc2p and Spc3p subunits. The yeast cells can also lack one or more functional subunits of the yeast signal peptidase complex and comprise eukaryotic homologues of the yeast Sec11p, Spc1p, Spc2p and Spc3p subunits. Methods for constructing these yeast cells as well as methods for production of heterologous proteins in these yeast cells are also provided.
2. Background Art
Signal peptidase complex (SPC) in the endoplasmic reticulum (ER) functions in the cleavage of N-terminal signal peptides from proteins that are in transit across the ER membrane (4,39). This almost immediate recognition of translocating polypeptides is critical in maintaining homeostasis in all eukaryotic cells. Despite the importance of the signal peptide cleavage reaction, surprisingly little is known concerning the function of each subunit. The partially purified SPC from yeast appears to contain four subunits, known as Sec11p, Spc1p, Spc2p and Spc3p (13,61) (Table 1).
Genetic and biochemical analyses have demonstrated that (i) Sec11p genetically interacts with Spc1p, Spc2p, and Spc3p; (ii) Sec11p is essential for growth, signal peptidase activity and protein degradation (6,33); (iii) a “core complex” containing Sec11p and Spc3p is sufficient for cell growth and signal sequence cleavage in vivo; and (iv) although not essential for cell growth, Spc1p is important for efficient signal peptidase activity and Spc2p is important for signal peptidase activity and cell viability at high temperatures.
Protein purification studies in other eukaryotic systems have shown that a multisubunit SPC is also present in canine cells (11). The complementary DNAs (cDNAs) encoding all five canine subunits have been cloned and sequenced (19,20,26,45,46). As shown in Table 2, the yeast subunit, Sec11p, is homologous to SPC18 and SPC21 of the mammalian SPC, yeast subunit Spc1p is homologous to mammalian SPC12, yeast subunit Spc2p is homologous to mammalian SPC25 and yeast subunit Spc3p is homologous to mammalian SPC22/23. The yeast and canine homologues display similarities in their sequences, isoelectric points, and hydropathy profiles as determined by the method of Kyte and Doolittle (30).
SPC purified from avian cells contains only two subunits (p19 and gp23), which are homologous to Sec11p and SPC22/23 (a glycoprotein), respectively (2,35). Unlike the situation in eukaryotes, bacterial leader peptidase contains a single polypeptide with a MW of 36 kDa (63). Leader peptidase is related to Sec11p, SPC18 and SPC21 in that these proteins contain three regions of sequence homology (3,7,54). Because leader peptidase catalyzes signal peptide cleavage as a monomer, these regions of homology may represent all or part of the catalytic site in eukaryotic signal peptidase. As used herein, “catalytic site” means the three regions (domains) of sequence homology which are recognized in the art to be the “consensus sequence” of a eukaryotic signal peptidase. Specifically, the consensus sequence is described in Dalby et al. as the three most highly conserved domains shown in FIG. 4 of Dalby et al.
The yeast
Saccharomyces cerevisiae
has become increasingly popular for the expression and secretion of foreign proteins in pharmaceutical and biotechnology industries for the following reasons: (i) yeast and humans possess similar cellular machinery for the expression and the proper modification of heterologous eukaryotic gene products; and (ii) yeast can be grown economically. However, processing of mammalian signal sequences in yeast is often inefficient. To solve this problem, yeast expression vectors containing one of several yeast signal sequences (e.g., &agr; factor, CPY, invertase, etc.) upstream of mammalian genes are currently being used to obtain more efficient processing. This approach has limited success due to the fact that the efficient processing of mature heterologous proteins by the yeast signal peptidase can be affected by sequences on the carboxyl side of the cleavage site (25).
The present invention overcomes these limitations by providing a yeast cell comprising eukaryotic signal peptidase subunits homologous to the yeast SPC subunits, that process mammalian signal sequences more efficiently than yeast SPC, thereby increasing production of the heterologous proteins in these genetically engineered cells. Thus, the yeast cells of the present invention are improved hosts for heterologous expression and secretion of mammalian proteins than currently available yeast strains.
SUMMARY OF THE INVENTION
The present invention provides a yeast cell comprising a nucleic acid functionally encoding a eukaryotic (e.g., mammalian) protein homologue of a subunit (Sec11p, Spc1p, Spc2p and/or Spc3p) of the yeast signal peptidase complex. The yeast cell of this invention can be, for example, of the genus Saccharomyces, Schizosaccharomyces, Pichia, Hansenula, Kluyveromyces and/or Yarrowia. Furthermore, the yeast cell can lack one or more functional subunits (Sec11p, Spc1p, Spc2p and/or Spc3p) of the yeast signal peptidase complex.
The present invention further provides a method for producing a protein heterologous to a yeast cell comprising expressing, in the yeast cell of this invention, a nucleic acid functionally encoding the heterologous protein under conditions which permit the expression of the nucleic acid as a precursor protein having a signal peptide and processing of the precursor protein to a signal peptide-cleaved form of the protein.
A method is also provided for obtaining increased production of a protein heterologous to a yeast cell comprising expressing, in the yeast cell of this invention, a nucleic acid functionally encoding the heterologous protein under conditions which permit the expression of the nucleic acid as a precursor protein having a signal peptide and processing of the precursor protein to a signal peptide-cleaved form of the protein.
Furthermore, the present invention provides a method for producing a yeast cell capable of producing a protein heterologous to the yeast cell, comprising introducing into the yeast cell (with or without one or more functional SPC subunits) a nucleic acid which functionally encodes one or more eukaryotic protein homologues of a subunit of the yeast signal peptidase complex under conditions permitting the expression of the nucleic acid as the protein homologues.
Additionally provided is a method for producing a yeast cell capable of increased production of a protein heterologous to the yeast cell, comprising introducing into the yeast cell (with or without one or more functional SPC subunits) a nucleic acid which functionally encodes one or more eukaryotic protein homologues of the signal peptidase complex under conditions permitting the expression of the nucleic acid as the protein homologues.
Various other objectives and advantages of the present invention will become apparent from the following description.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention may be understood more readily by reference to the following detailed description of specific embodiments and the Examples included herein. As used in the claims, “a” can include multiples.
The present invention provides a yeast cell comprising a nucleic acid The subunit of the SPC can be Sec11p, Spc1p, Spc2p and/or Spc3p. The yeast cell of this invention can comprise a nucleic acid encoding a subunit homologue of the yeast SPC subunit from, for example, mammals, birds, fish, reptiles, amphibians, arthropods, plants, fungi and other eukaryotic organisms possessing a multisubunit SPC. The yeast cell comprising a nucleic acid encoding a subunit homologue of the yeast SPC subunit can contain all of the yeast SPC subunits in functional form or t
Fang Hong
Green Neil
Kaer Luc Van
McKelvey Terry
Needle & Rosenberg P.C.
Vanderbilt University
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