Markku Saloheimo - Academia.edu (original) (raw)

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Papers by Markku Saloheimo

Research paper thumbnail of Monitoring of transcript regulation and protein production related stress responses in Pichia pastoris secreting Fab antibody fragments

Microbial Cell Factories, 2006

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Research paper thumbnail of Optimization of cDNA-AFLP experiments using genomic sequence data

Bioinformatics, 2005

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Research paper thumbnail of Inducing an elevated unfolded protein response by using an isolated heterologous nucleic acid endocing a HAC1 protein, a PTC2 protein or an 1re1 protein

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Research paper thumbnail of Novel xylanase from trichoderma reesei, method for production thereof, and methods employing this enzyme

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Research paper thumbnail of Trichoderma Reesei Swollenin Protein and Encoding Dna Sequence

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Research paper thumbnail of Use of a growth-associated control algorithm for efficient production of a heterologous laccase in Trichoderma reesei in fed-batch and continuous cultivation

Enzyme and Microbial Technology, Sep 1, 2007

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Research paper thumbnail of Novel Microbial Enzymes and Their Use

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Research paper thumbnail of Method for production of secreted proteins in fungi

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Research paper thumbnail of Novel laccase enzyme and the gene encoding the enzyme

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Research paper thumbnail of Production of fucosylated glycoproteins

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Research paper thumbnail of Increased production of secreted proteins by recombinant eukaryotic cells

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Research paper thumbnail of Proteins having tyrosinase activity

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Research paper thumbnail of O-Mannosyltransferase deficient filamentous fungal cells and methods of use thereof

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Research paper thumbnail of Multiple proteases deficient filamentous fungal cells and methods of use thereof

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Research paper thumbnail of Protease Deficient Filamentous Fungal Cells And Methods of Use Thereof

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Research paper thumbnail of Enabling Low Cost Biopharmaceuticals: A Systematic Approach to Delete Proteases from a Well-Known Protein Production Host Trichoderma reesei

PLOS ONE, 2015

The filamentous fungus Trichoderma reesei has tremendous capability to secrete proteins. Therefor... more The filamentous fungus Trichoderma reesei has tremendous capability to secrete proteins. Therefore, it would be an excellent host for producing high levels of therapeutic proteins at low cost. Developing a filamentous fungus to produce sensitive therapeutic proteins requires that protease secretion is drastically reduced. We have identified 13 major secreted proteases that are related to degradation of therapeutic antibodies, interferon alpha 2b, and insulin like growth factor. The major proteases observed were aspartic, glutamic, subtilisin-like, and trypsin-like proteases. The seven most problematic proteases were sequentially removed from a strain to develop it for producing therapeutic proteins. After this the protease activity in the supernatant was dramatically reduced down to 4% of the original level based upon a casein substrate. When antibody was incubated in the six protease deletion strain supernatant, the heavy chain remained fully intact and no degradation products were observed. Interferon alpha 2b and insulin like growth factor were less stable in the same supernatant, but full length proteins remained when incubated overnight, in contrast to the original strain. As additional benefits, the multiple protease deletions have led to faster strain growth and higher levels of total protein in the culture supernatant.

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Research paper thumbnail of Monitoring of transcript regulation and protein production related stress responses in Pichia pastoris secreting Fab antibody fragments

Microbial Cell Factories, 2006

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Research paper thumbnail of Genome-wide analysis of protein production physiology in the filamentous fungus Trichoderma reesei

Microbial Cell Factories

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Research paper thumbnail of Comparison of intracellular and secretion-based strategies for production of human α-galactosidase A in the filamentous fungus Trichoderma reesei

BMC biotechnology, 2014

Trichoderma reesei is known as a good producer of industrial proteins but has hitherto been less ... more Trichoderma reesei is known as a good producer of industrial proteins but has hitherto been less successful in the production of therapeutic proteins. In order to elucidate the bottlenecks of heterologous protein production, human α-galactosidase A (GLA) was chosen as a model therapeutic protein. Fusion partners were designed to compare the effects of secretion using a cellobiohydrolase I (CBHI) carrier and intracellular production using a gamma zein peptide from maize (ZERA) which accumulates inside the endoplasmic reticulum (ER). The two strategies were compared on the basis of expression levels, purification performance, enzymatic activity, bioreactor cultivations, and transcriptional profiling. Constructs were cloned into the cbh1 locus of the T. reesei strain Rut-C30. The secretion and intracellular strains produced 20 mg/l and 636 mg/l of GLA respectively. Purifications of secreted product were accomplished using Step-Tactin affinity columns and for intracellular product, a me...

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Research paper thumbnail of Expression of the Trichoderma reesei tyrosinase 2 in Pichia pastoris: isotopic labeling and physicochemical characterization

Protein expression and purification, 2007

Trichoderma reesei tyrosinase TYR2 has been demonstrated to be able to oxidize various phenolic c... more Trichoderma reesei tyrosinase TYR2 has been demonstrated to be able to oxidize various phenolic compounds and also peptide and protein bound tyrosine, and thus is of great interest for different biotechnological applications. In order to understand the reaction mechanism of the enzyme it would be essential to solve its three dimensional structure. Pichia pastoris is a suitable expression system for the production of recombinant enzymes for NMR studies and therefore we expressed TYR2 in this host. As a result of extensive optimization, the production yield of active histidine tagged tyrosinase purified from P. pastoris shake flask cultures was increased from 2.5 to 24 mg/L. Correct copper concentration in the growth medium was critical for the expression of this copper containing enzyme. Our analysis showed that TYR2 expressed in P. pastoris is post-translationally modified; the C-terminal domain of 153 amino acids of the protein is proteolytically cleaved off from the catalytic doma...

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Research paper thumbnail of Monitoring of transcript regulation and protein production related stress responses in Pichia pastoris secreting Fab antibody fragments

Microbial Cell Factories, 2006

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Research paper thumbnail of Optimization of cDNA-AFLP experiments using genomic sequence data

Bioinformatics, 2005

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Research paper thumbnail of Inducing an elevated unfolded protein response by using an isolated heterologous nucleic acid endocing a HAC1 protein, a PTC2 protein or an 1re1 protein

Bookmarks Related papers MentionsView impact

Research paper thumbnail of Novel xylanase from trichoderma reesei, method for production thereof, and methods employing this enzyme

Bookmarks Related papers MentionsView impact

Research paper thumbnail of Trichoderma Reesei Swollenin Protein and Encoding Dna Sequence

Bookmarks Related papers MentionsView impact

Research paper thumbnail of Use of a growth-associated control algorithm for efficient production of a heterologous laccase in Trichoderma reesei in fed-batch and continuous cultivation

Enzyme and Microbial Technology, Sep 1, 2007

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Research paper thumbnail of Novel Microbial Enzymes and Their Use

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Research paper thumbnail of Method for production of secreted proteins in fungi

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Research paper thumbnail of Novel laccase enzyme and the gene encoding the enzyme

Bookmarks Related papers MentionsView impact

Research paper thumbnail of Production of fucosylated glycoproteins

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Research paper thumbnail of Increased production of secreted proteins by recombinant eukaryotic cells

Bookmarks Related papers MentionsView impact

Research paper thumbnail of Proteins having tyrosinase activity

Bookmarks Related papers MentionsView impact

Research paper thumbnail of O-Mannosyltransferase deficient filamentous fungal cells and methods of use thereof

Bookmarks Related papers MentionsView impact

Research paper thumbnail of Multiple proteases deficient filamentous fungal cells and methods of use thereof

Bookmarks Related papers MentionsView impact

Research paper thumbnail of Protease Deficient Filamentous Fungal Cells And Methods of Use Thereof

Bookmarks Related papers MentionsView impact

Research paper thumbnail of Enabling Low Cost Biopharmaceuticals: A Systematic Approach to Delete Proteases from a Well-Known Protein Production Host Trichoderma reesei

PLOS ONE, 2015

The filamentous fungus Trichoderma reesei has tremendous capability to secrete proteins. Therefor... more The filamentous fungus Trichoderma reesei has tremendous capability to secrete proteins. Therefore, it would be an excellent host for producing high levels of therapeutic proteins at low cost. Developing a filamentous fungus to produce sensitive therapeutic proteins requires that protease secretion is drastically reduced. We have identified 13 major secreted proteases that are related to degradation of therapeutic antibodies, interferon alpha 2b, and insulin like growth factor. The major proteases observed were aspartic, glutamic, subtilisin-like, and trypsin-like proteases. The seven most problematic proteases were sequentially removed from a strain to develop it for producing therapeutic proteins. After this the protease activity in the supernatant was dramatically reduced down to 4% of the original level based upon a casein substrate. When antibody was incubated in the six protease deletion strain supernatant, the heavy chain remained fully intact and no degradation products were observed. Interferon alpha 2b and insulin like growth factor were less stable in the same supernatant, but full length proteins remained when incubated overnight, in contrast to the original strain. As additional benefits, the multiple protease deletions have led to faster strain growth and higher levels of total protein in the culture supernatant.

Bookmarks Related papers MentionsView impact

Research paper thumbnail of Monitoring of transcript regulation and protein production related stress responses in Pichia pastoris secreting Fab antibody fragments

Microbial Cell Factories, 2006

Bookmarks Related papers MentionsView impact

Research paper thumbnail of Genome-wide analysis of protein production physiology in the filamentous fungus Trichoderma reesei

Microbial Cell Factories

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Research paper thumbnail of Comparison of intracellular and secretion-based strategies for production of human α-galactosidase A in the filamentous fungus Trichoderma reesei

BMC biotechnology, 2014

Trichoderma reesei is known as a good producer of industrial proteins but has hitherto been less ... more Trichoderma reesei is known as a good producer of industrial proteins but has hitherto been less successful in the production of therapeutic proteins. In order to elucidate the bottlenecks of heterologous protein production, human α-galactosidase A (GLA) was chosen as a model therapeutic protein. Fusion partners were designed to compare the effects of secretion using a cellobiohydrolase I (CBHI) carrier and intracellular production using a gamma zein peptide from maize (ZERA) which accumulates inside the endoplasmic reticulum (ER). The two strategies were compared on the basis of expression levels, purification performance, enzymatic activity, bioreactor cultivations, and transcriptional profiling. Constructs were cloned into the cbh1 locus of the T. reesei strain Rut-C30. The secretion and intracellular strains produced 20 mg/l and 636 mg/l of GLA respectively. Purifications of secreted product were accomplished using Step-Tactin affinity columns and for intracellular product, a me...

Bookmarks Related papers MentionsView impact

Research paper thumbnail of Expression of the Trichoderma reesei tyrosinase 2 in Pichia pastoris: isotopic labeling and physicochemical characterization

Protein expression and purification, 2007

Trichoderma reesei tyrosinase TYR2 has been demonstrated to be able to oxidize various phenolic c... more Trichoderma reesei tyrosinase TYR2 has been demonstrated to be able to oxidize various phenolic compounds and also peptide and protein bound tyrosine, and thus is of great interest for different biotechnological applications. In order to understand the reaction mechanism of the enzyme it would be essential to solve its three dimensional structure. Pichia pastoris is a suitable expression system for the production of recombinant enzymes for NMR studies and therefore we expressed TYR2 in this host. As a result of extensive optimization, the production yield of active histidine tagged tyrosinase purified from P. pastoris shake flask cultures was increased from 2.5 to 24 mg/L. Correct copper concentration in the growth medium was critical for the expression of this copper containing enzyme. Our analysis showed that TYR2 expressed in P. pastoris is post-translationally modified; the C-terminal domain of 153 amino acids of the protein is proteolytically cleaved off from the catalytic doma...

Bookmarks Related papers MentionsView impact