Abstract: A method of producing a protein of interest (POI) by culturing a recombinant eukaryotic cell line comprising an expression construct comprising a regulatable promoter and a nucleic acid molecule encoding a POI under the transcriptional control of said promoter, comprising the steps a) cultivating the cell line with a basal carbon source repressing the promoter, b) cultivating the cell line with a limited amount of a supplemental carbon source de-repressing the promoter to induce production of the POI at a transcription rate of at least 15% as compared to the native pGAP promoter, and c) producing and recovering the POI; and further an isolated regulatable promoter and a respective expression system.

Abstract: A method of producing a protein of interest (POI) by culturing a recombinant eukaryotic cell line comprising an expression construct comprising a regulatable promoter and a nucleic acid molecule encoding a POI under the transcriptional control of said promoter, comprising the steps a) cultivating the cell line with a basal carbon source repressing the promoter, b) cultivating the cell line with a limited amount of a supplemental carbon source de-repressing the promoter to induce production of the POI at a transcription rate or at least 15% as compared to the native pGAP promoter, and c) producing and recovering the POI; and further an isolated regulatable promoter and a respective expression system.

Abstract: An isolated nucleic acid encoding a leader, which has a specific sequence, an isolated leader peptide encoded by such nucleic acid, an expression cassette comprising such nucleic acid encoding a leader operably linked to a nucleic acid sequence encoding a POI, a recombinant yeast host cell or a vector comprising such expression cassette, a method of producing a POI in such yeast host cell, and further the use of the specific nucleic acid for the secretion of a POI from a host cell and/or to increase the secretion of a POI from a host cell.

Abstract: An isolated and/or artificial pG1-x promoter, which is a functional variant of the carbon source regulatable pG1 promoter of Pichia pastoris identified by SEQ ID 1, which pG1-x promoter consists of or comprises at least a part of SEQ ID 1 with a length of at least 293 bp, characterized by the following promoter regions: a) at least one core regulatory region comprising the nucleotide sequences SEQ ID 2 and SEQ ID 3; and b) a non-core regulatory region, which is any region within the pG1-x promoter sequence other than the core regulatory region; wherein the pG1-x promoter comprises at least one mutation in any of the promoter regions and a sequence identity of at least 80% in SEQ ID 2 and SEQ ID 3, and a sequence identity of at least 50% in any region other than SEQ ID 2 or SEQ ID 3; and further wherein the pG1-x promoter is characterized by the same or an increased promoter strength and induction ratio as compared to the pG1 promoter, wherein the promoter strength is at least 1.

Abstract: An isolated nucleic acid encoding a leader, which has a specific sequence, an isolated leader peptide encoded by such nucleic acid, an expression cassette comprising such nucleic acid encoding a leader operably linked to a nucleic acid sequence encoding a POI, a recombinant yeast host cell or a vector comprising such expression cassette, a method of producing a POI in such yeast host cell, and further the use of the specific nucleic acid for the secretion of a POI from a host cell and/or to increase the secretion of a POI from a host cell.

Abstract: The present invention is in the field of recombinant biotechnology, in particular in the field of protein expression. The invention generally relates to a method of expressing a protein of interest (POI) from a host cell. The invention relates particularly to improving a host cell's capacity to express and/or secrete a protein of interest and use of the host cell for protein expression. The invention also relates to cell culture technology, and more specifically to culturing cells to produce desired molecules for medical purposes or food products.

Abstract: A method of producing a protein of interest (POI) by culturing a recombinant eukaryotic cell line comprising an expression construct comprising a regulatable promoter and a nucleic acid molecule encoding a POI under the transcriptional control of said promoter, comprising the steps a) cultivating the cell line with a basal carbon source repressing the promoter, b) cultivating the cell line with a limited amount of a supplemental carbon source de-repressing the promoter to induce production of the POI at a transcription rate or at least 15% as compared to the native pGAP promoter, and c) producing and recovering the POI; and further an isolated regulatable promoter and a respective expression system.

Abstract: The present invention is in the field of recombinant biotechnology, in particular in the field of protein expression. The invention generally relates to a method of expressing a protein of interest (POI) from a host cell. The invention relates particularly to improving a host cell's capacity to express and/or secrete a protein of interest and use of the host cell for protein expression. The invention also relates to cell culture technology, and more specifically to culturing cells to produce desired molecules for medical purposes or food products.

Abstract: A method of producing a protein of interest (POI) by culturing a recombinant eukaryotic cell line comprising an expression construct comprising a regulatable promoter and a nucleic acid molecule encoding a POI under the transcriptional control of said promoter, comprising the steps a) cultivating the cell line with a basal carbon source repressing the promoter, b) cultivating the cell line with a limited amount of a supplemental carbon source de-repressing the promoter to induce production of the POI at a transcription rate of at least 15% as compared to the native pGAP promoter, and c) producing and recovering the POI; and further an isolated regulatable promoter and a respective expression system.

Abstract: The invention relates to an isolated nucleic acid sequence comprising a promoter, which is a native sequence of Pichia pastoris comprising the nucleic acid sequence of pCS1 of SEQ ID 1, or a functionally active variant thereof which is a size variant, a mutant or hybrid of SEQ ID 1, or a combination thereof, expression constructs and recombinant host cells comprising the promoter, and a method of producing a protein of interest under the control of the promoter. It further relates to a method to identify a constitutive promoter from eukaryotic cells, and an isolated nucleic acid sequence comprising a promoter which when operatively linked to a nucleotide sequence encoding a protein of interest directs the expression thereof in a host cell at an expression level that is higher than under control of the native pGAP promoter at high and low growth rates.

Abstract: The invention relates to an isolated nucleic acid sequence comprising a promoter, which is a native sequence of Pichia pastoris comprising the nucleic acid sequence of pCS1 of SEQ ID NO:1, or a functionally active variant thereof which is a size variant, a mutant or hybrid of SEQ ID NO:1, or a combination thereof, expression constructs and recombinant host cells comprising the promoter, and a method of producing a protein of interest under the control of the promoter. It further relates to a method to identify a constitutive promoter from eukaryotic cells, and an isolated nucleic acid sequence comprising a promoter which when operatively linked to a nucleotide sequence encoding a protein of interest directs the expression thereof in a host cell at an expression level that is higher than under control of the native pGAP promoter at high and low growth rates.

Abstract: An isolated nucleic acid encoding a leader, which has a specific sequence, an isolated leader peptide encoded by such nucleic acid, an expression cassette comprising such nucleic acid encoding a leader operably linked to a nucleic acid sequence encoding a POI, a recombinant yeast host cell or a vector comprising such expression cassette, a method of producing a POI in such yeast host cell, and further the use of the specific nucleic acid for the secretion of a POI from a host cell and/or to increase the secretion of a POI from a host cell.

Abstract: The invention refers to a method of producing a recombinant polypeptide of interest (POI) in a cell culture, comprising genetically engineering a eukaryotic cell line—to specifically cause prolongation of the G2+M cell cycle phase in a pre-culture phase, and—to produce the POI in a producing phase following the pre-culture phase, a high producer cell line and cell culture as well as a method of increasing the yield of a recombinant POI production in a cell culture.

Abstract: The invention relates to an isolated nucleic acid sequence comprising a promoter, which is a native sequence of Pichia pastoris comprising the nucleic acid sequence of pCS1 of SEQ ID NO:1, or a functionally active variant thereof which is a size variant, a mutant or hybrid of SEQ ID NO:1, or a combination thereof, expression constructs and recombinant host cells comprising the promoter, and a method of producing a protein of interest under the control of the promoter. It further relates to a method to identify a constitutive promoter from eukaryotic cells, and an isolated nucleic acid sequence comprising a promoter which when operatively linked to a nucleotide sequence encoding a protein of interest directs the expression thereof in a host cell at an expression level that is higher than under control of the native pGAP promoter at high and low growth rates.

Abstract: A method of producing a protein of interest (POI) by culturing a recombinant eukaryotic cell line comprising an expression construct comprising a regulatable promoter and a nucleic acid molecule encoding a POI under the transcriptional control of said promoter, comprising the steps a) cultivating the cell line with a basal carbon source repressing the promoter, b) cultivating the cell line with a limited amount of a supplemental carbon source de-repressing the promoter to induce production of the POI at a transcription rate of at least 15% as compared to the native pGAP promoter, and c) producing and recovering the POI; and further an isolated regulatable promoter and a respective expression system.

Abstract: The invention refers to a method of producing a recombinant polypeptide of interest (POI) in a cell culture, comprising genetically engineering a eukaryotic cell line—to specifically cause prolongation of the G2+M cell cycle phase in a pre-culture phase, and—to produce the POI in a producing phase following the pre-culture phase, a high producer cell line and cell culture as well as a method of increasing the yield of a recombinant POI production in a cell culture.

Abstract: The present invention relates to methods for increasing the secretion of a protein of interest (POI) from a eukaryotic cell comprising co-expression of a POI and of at least one protein that enhances protein secretion, said enhancing protein being selected from the group consisting of BMH2, BFR2, C0G6, C0Y1, CUP5, IMH 1, KIN2, SEC31, SSA4 and SSE1. The invention further relates to a yeast promoter sequence, in particular to a promoter sequence of the PET9 gene of P. pastoris, having, under comparable conditions, an increased promoter activity relative to a promoter sequence of the GAP protein. The invention further relates to an expression vector comprising such a promoter sequence and to the use of such an expression vector for expression of a POI in a host cell. The invention further relates to new yeast promoter sequences of genes from P. pastoris, which are useful for expression of a POI in yeast.