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
1999-12-30
2002-02-05
Schwartzman, Robert A. (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...
C435S254210
Reexamination Certificate
active
06344341
ABSTRACT:
This application is the national phase under 35 U.S.C. §371 of PCT International Application No. PCT/FI98/00576 which has an International filing date of Jul. 8, 1998 which designated the United States of America.
FIELD OF THE INVENTION
This invention relates to recombinant-DNA-technology. Specifically this invention relates to new recombinant yeast cells transformed with SEB1 gene or its homologs. A yeast cell transformed with several copies of a SEB1 gene or a gene homologous to SEB1 has an increased capacity to produce secreted foreign or endogenous proteins.
Further, said new recombinant yeast cells, when transformed with genes expressing suitable hydrolytic enzymes can hydrolyze and/or utilize appropriate macromolecular/polymeric compounds more efficiently, which results in increased cell mass production and/or more versatile utilization of the compounds in relevant biotechnical applications.
BACKGROUND OF THE INVENTION
The development of recombinant DNA methods has made it possible to produce proteins in heterologous host systems. This possibility greatly facilitates production of e.g. proteins of therapeutic importance which normally occur in nature in very low amounts or are otherwise difficult to isolate or purify. Such proteins include growth factors, hormones and other biologically active proteins or peptides which traditionally have been isolated from human or animal tissues or body fluids e.g. blood serum or urine. The increasing danger of the presence of human pathogenic viruses such as HBV, HIV, and oncogenic viruses, prions, or other pathogens in the human or animal tissues or body fluids has greatly speeded up the search for heterologous production systems for these therapeutics. Other proteins of clinical importance are viral or other microbial or human parasite proteins needed for diagnostics and for vaccines especially of such organisms which are difficult to grow in vitro or in tissue culture, or are dangerous human pathogens. These include viruses like HBV, HIV, yellow fever, rubella, FMDV, rabies, and human parasites such as
Plasmodium falciparum
causing malaria.
A further group of proteins for which heterologous production systems have been or are being developed are secreted enzymes, especially those hydrolyzing plant material, and which are needed in food and fodder production as well as in other industrial processes including textile industry and pulp and paper industry. The possibility of producing proteins in heterologous systems or production of endogenous proteins in genetically engineered cells increases their yields and greatly facilitates their purification and has already by now had a great impact on studies of structure and function of many important enzymes and other proteins. The production and secretion of foreign hydrolytic enzymes in yeast for example, results in improvements in processes based on industrial yeast strains such as distiller's, brewer's or baker's yeasts.
Various production systems have been and are being developed including bacteria, yeasts, filamentous fungi, animal and plant cell cultures and even multicellular organisms like transgenic animals and plants. All of these different systems have their advantages, even if disadvantages, and all of them are needed.
The yeast
Saccharomyces cerevisiae
is at the moment the best known eukaryote at genetic level. Its whole genome sequence became public in data bases on Apr. 24, 1996. As a eukaryotic microbe it possesses the advantages of a eukaryotic cell like most if not all of the post-translational modifications of eukaryotes, and as a microbe it shares the easy handling and cultivation properties of bacteria. The large scale fermentation systems are well developed for
S. cerevisiae
which has a long history as a work horse of biotechnology including production of food ingredients and beverages such as beer and wine.
The yeast genetic methods are by far the best developed among eukaryotes based on the vast knowledge obtained by classical genetics. This made it easy to adopt and further develop for yeast the gene technology procedures first described for
Escherichia coli.
Along other lines the methods for constructing yeast strains producing foreign proteins have been developed to a great extent (Romanos et. al., 1992).
Secretion of the proteins into the culture medium involves transfer of the proteins through the various membrane enclosed compartments constituting the secretory pathway. First the proteins are translocated into the lumen of the endoplasmic reticulum, ER. From there on the proteins are transported in membrane vesicles to the Golgi complex and from Golgi to plasma membrane. The secretory process involves several steps in which vesicles containing the secreted proteins are pinched off from the donor membrane, targetted to and fused with the acceptor membrane. At each of these steps function of several different proteins are needed.
The yeast secretory pathway and a great number of genes involved in it have been elucidated by isolation of conditional lethal mutants deficient in certain steps of the secretory process (Novick et al., 1980; 1981). Mutation in a protein, needed for a particular transfer step results in accumulation of the secreted proteins in the preceding membrane compartment. Thus proteins can accumulate in the cytoplasm, at ER, Golgi or in vesicles between ER and Golgi, or in vesicles between Golgi and plasma membrane.
More detailed analysis of the genes and proteins involved in the secretory process has become possible upon cloning the genes and characterization of the function of their encoded proteins. A picture is emerging which indicates that in all steps several interacting proteins are functioning. The number of genes is rapidly increasing that are involved in protein secretion and that were first identified in and isolated from
S. cerevisiae
and were later found in other organisms including lower and higher eukaryotes. The structural and functional homology has been shown for many of such proteins.
We have recently cloned a new yeast gene, SEB1 (Toikkanen et al., 1996) which encodes the &bgr;-subunit of the trimeric Sec61 complex (hence the name: SEB=SEc61 Beta subunit) that is likely to represent the protein conducting channel of the ER both in co- and post-translational translocation (Hanein et al., 1996). In the former it functions in close connection with the ribosome and in latter it forms a heptameric membrane protein complex with the tetrameric Sec62/Sec63 complex (Panzner et al., 1995). Genes with sequence similarity to the SEB1 gene are found in plant and mammalian cells indicating that the Sec61 translocation complex is conserved in evolution. In fact, similar components function in protein translocation also in prokaryotes (discussed in Toikkanen et al., 1996). This further supports the conserved and central role of the SEB1 gene in protein secretion and intracellular transport. However, no reports exist so far on any positive effect of the SEB1 or its homologs in other yeasts, plant or animal cells on secretion when overexpressed, which effect we are showing in this invention for the yeast SEB1 gene. It should be noticed that Seb1 protein is present in a different protein complex and at different location than the Sso proteins which we have previously shown to enhance production of secreted proteins when present in the cells in higher than normal amounts.
Knowledge on the protein secretion process in
S. cerevisiae
is rapidly increasing. Less is known about the secretory system of other yeasts such as Kluyveromyces, Schizosaccharomyces, Pichia and Hansenula which, however, have proven useful hosts for production of foreign proteins (Buckholz and Gleeson, 1991; Romanos et al., 1992), or Candida and Yarrowia which also are interesting as host systems. The genetics and molecular biology of these yeasts are not as developed as for Saccharomyces but the advantages of these yeasts as production hosts are the same as for Saccharomyces.
Several attempts have been made and published previously to increase
Keränen Sirkka
Söderlund Hans
Tieaho Ville
Toikkanen Jaana
Birch, Stewart, Kolasch & Birch, Ltd.
Davis Katharine F
Schwartzman Robert A.
Valtion teknillinen tetkimuskeskus
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