Method for production of a heterologous protein using yeast-type host cells
Inventors
Gellissen, Gerd • Klabunde, Jens • Hollenberg, Cornelis • Degelmann, Adelheid
Assignees
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Publication Number
US-7858343-B2
Publication Date
2010-12-28
Expiration Date
Abstract
A heterologous protein is produced by a method in which yeast-type host cells are prepared, which, in addition to the at least one endogenous homologous DNA sequence coding for a first calnexin, includes at least one recombinant DNA sequence coding for the protein and at least one additional recombinant DNA sequence coding for at least one second calnexin. The protein in the host cell represents a heterologous secretable protein. The host cells are induced to express the heterologous protein via the at least one sequence coding for the protein and to overexpress calnexins by the at least one additional sequence coding for the at least one second calnexin. The host cells are then induced to secrete the heterologous protein. The secreted heterologous protein is then separated off.
Core Innovation
The invention relates to a method for producing a heterologous, secretable protein in yeast-type host cells by using recombinant DNA to express the protein while simultaneously overexpressing calnexins. The host cells contain at least one own, homologous DNA sequence coding for at least a first calnexin and at least one additional recombinant DNA sequence coding for a second calnexin. The heterologous protein is expressed in the host cell and then secreted and separated.
A central aspect is causing the host cells to express the heterologous protein and, with at least one additional sequence coding for a second calnexin, to overexpress calnexins, followed by secretion and separating the secreted heterologous protein. The approach is framed around increasing secretion by calnexin overexpression rather than deactivating calnexin, and is presented as surprising versus prior approaches.
The disclosure emphasizes yeast types and configurations for recombinant expression, including yeast genera selected from Saccharomyces, Schizosaccharomyces, Kluyveromyces, Hansenula, Pichia, Arxula, Schwanniomyces, Candida, and Yarrowia. Preferred selections are Hansenula and Pichia, in particular Hansenula polymorpha, and the calnexin overexpression is implemented using recombinant DNA encoding at least a first endogenous calnexin and an additional second calnexin.
Claims Coverage
The document provides one independent claim directed to producing a heterologous, secretable protein in yeast host cells by co-expressing a heterologous protein construct and an additional second calnexin construct, followed by secretion and separating the secreted protein. The dependent claims refine this framework by narrowing yeast genera or species, specifying recombinant DNA insertion and integration, defining relationships between calnexins, and constraining the heterologous protein to particular forms, including post-translational glycosylation.
Yeast host cells with endogenous first calnexin and recombinant second calnexin
Preparing host cells from at least one yeast type of genera Saccharomyces, Schizosaccharomyces, Kluyveromyces, Hansenula, Pichia, Arxula, Schwanniomyces, Candida, and Yarrowia, which along with their own homologous DNA sequence coding for at least a first calnexin also contain at least one recombinant DNA sequence coding for the protein and at least one additional recombinant DNA sequence coding for a second calnexin, wherein the protein in the host cell represents a heterologous, secretable protein.
Co-expression of heterologous secretable protein and calnexins via recombinant DNA
Causing the host cells, with the help of at least one sequence coding for the protein, to express the heterologous protein, and, with at least one additional sequence coding for a second calnexin, to overexpress calnexins.
Secretion and separation of the heterologous protein
Causing the host cells to secrete the heterologous protein, and separating the secreted heterologous protein.
Methylothrophenic yeast selection
Selecting the methylothrophenic yeast from the genera Hansenula and Pichia.
Simultaneous insertion of recombinant DNA for protein and second calnexin
Preparing the host cell by simultaneously inserting recombinant DNA sequences encoding a heterologous protein and at least a second calnexin into the host cell.
Genomic integration of vectors
Integrating the vectors into the genome of the cell.
Structurally similar calnexins
The first and second calnexins are at least structurally similar.
Post-translational glycosylation of the heterologous protein
The heterologous protein is a post-translationally glycosylated protein.
Overall, claim coverage centers on a yeast-based production method where a heterologous, secretable protein is expressed while an additional second calnexin is overexpressed, followed by secretion and separation. Dependent claims further restrict the yeast genus, constrain recombinant DNA insertion and integration configurations, define relationships between first and second calnexins, and narrow the heterologous protein to post-translationally glycosylated proteins.
Stated Advantages
Increased secretion of heterologous secretable proteins by overexpressing calnexins.
Secreted protein quantity is increased in the examples without gene dosage changes.
Documented Applications
Production of heterologous secretable proteins in yeast-type host cells, including glycosylated heterologous proteins exemplified by alginate C5 epimerase, consensus phytase, γ-interferon, and serum albumin.
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