Method for rapidly screening microbial hosts to identify certain strains with improved yield and/or quality in the expression of heterologous proteins
First Claim
1. A method for identifying an optimal population of Pseudomonas fluorescens (P. fluorescens) host cells for expression of at least one heterologous protein of interest comprising:
- a) assembling a strain array comprising at least four populations of P. fluorescens host cells, wherein each population of P. fluorescens host cells in the strain array is selected from the group consisting of;
i) a population of P. fluorescens host cells that has been genetically modified to reduce the expression of at least one target gene encoding a protease, wherein the protease is an aminopeptidase, a dipeptidase, a dipeptidyl-peptidase, a tripeptidyl peptidase, a peptidyl-dipeptidase, a serine-type carboxypeptidase, a metallocarboxypeptidase, a cysteine-type carboxypeptidase, an omegapeptidase, a serine proteinase, a threonine proteinase, a cysteine proteinase, an aspartic proteinase, or a metalloproteinase;
ii) a population of P. fluorescens host cells that has been genetically modified to increase the expression of at least one target gene encoding a protein that modulates protein processing, folding or translocation, wherein the protein that modulates protein processing, folding or translocation is a chaperone, a disulfide bond isomerase, a peptidyl-prolyl cis-trans isomerase, a GroES/EL folding modulator, a DnaKJ folding modulator, a Clp folding modulator, an Hsp90 folding modulator, a SecB folding modulator, a PapD chaperone, an HSP70 protein, an HSP110/SSE protein, an HSP40 (DNAJ-related) protein, a GRPE-like protein, an HSP90 protein, a CPN60 protein, a CPN10 protein, an HSP100 protein, a small HSP, a calnexin, a calreticulin, a PDI-related protein, a thioredoxin-related protein, a cyclophilin PPIase, a FK-506 binding protein, or parvulin PPIase; and
,iii) a population of P. fluorescens host cells that has been genetically modified to reduce the expression of at least one target gene encoding a protease, wherein the protease is an aminopeptidase, a dipeptidase, a dipeptidyl-peptidase, a tripeptidyl peptidase, a peptidyl-dipeptidase, a serine-type carboxypeptidase, a metallocarboxypeptidase, a cysteine-type carboxypeptidase, an omegapeptidase, a serine proteinase, a threonine proteinase, a cysteine proteinase, an aspartic proteinase, or a metalloproteinase, and to increase the expression of at least one target gene encoding a protein that modulates protein processing, folding or translocation, wherein the protein that modulates protein processing, folding or translocation is a chaperone, a disulfide bond isomerase, a peptidyl-prolyl cis-trans isomerase, a GroES/EL folding modulator, a DnaKJ folding modulator, a Clp folding modulator, an Hsp90 folding modulator, a SecB folding modulator, a PapD chaperone, an HSP70 protein, an HSP110/SSE protein, an HSP40 (DNAJ-related) protein, a GRPE-like protein, an HSP90 protein, a CPN60 protein, a CPN10 protein, an HSP100 protein, a small HSP, a calnexin, a calreticulin, a PDI-related protein, a thioredoxin-related protein, a cyclophilin PPIase, a FK-506 binding protein, or parvulin PPIase;
wherein each population of P. fluorescens host cells in the strain array is non-identical and wherein each population of P. fluorescens host cells in the strain array is physically separate one from another;
b) introducing into at least one cell of each of the at least four populations of P. fluorescens host cells in the strain array an expression cassette comprising at least one gene encoding the at least one heterologous protein of interest,wherein when the population of P. fluorescens host cells into which the expression cassette is introduced has been genetically modified to reduce the expression of at least one target gene encoding a protease as set forth in (i), the gene encoding the heterologous protein of interest does not encode the protease of (i),and wherein when the population of P. fluorescens host cells into which the expression cassette is introduced has been genetically modified to increase the expression of at least one target gene encoding a protein that modulates protein processing, folding or translocation as set forth in (ii), the gene encoding the heterologous protein of interest does not encode the protein that modulates protein processing, folding or translocation of (ii),and wherein when the population of P. fluorescens host cells has been genetically modified to reduce the expression of at least one target gene encoding a protease, and to increase the expression of at least one target gene encoding a protein that modulates protein processing, folding or translocation as set forth in (iii), the gene encoding the heterologous protein of interest does not encode the protease of (i) or the protein that modulates protein processing, folding or translocation of (ii);
c) maintaining the populations of P. fluorescens host cells in the strain array under conditions sufficient for the expression of said heterologous protein of interest encoded by the gene encoding the at least one heterologous protein of interest, in the at least four populations of P. fluorescens host cells in the strain array, and expressing said heterologous protein of interest encoded by the gene encoding the at least one heterologous protein of interest, in the at least four populations of P. fluorescens host cells in the strain array; and
,d) selecting the optimal population of P. fluorescens host cells in the strain array in which the heterologous protein of interest encoded by the gene encoding the at least one heterologous protein of interest is produced, by simultaneous screening of the at least four populations of P. fluorescens host cells in the strain array;
wherein the heterologous protein of interest encoded by the gene encoding the at least one heterologous protein of interest exhibits one or more of improved expression, improved activity, improved solubility, improved translocation, or reduced proteolytic degradation in the optimal population of P. fluorescens host cells compared to other populations of P. fluorescens host cells in the strain array.
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Abstract
The present invention provides an array for rapidly identifying a host cell population capable of producing a heterologous protein with improved yield and/or quality. The array comprises one or more host cell populations that have been genetically modified to increase the expression of one or more target genes involved in protein production, decrease the expression of one or more target genes involved in protein degradation, or both. One or more of the strains in the array may express the heterologous protein of interest in a periplasm compartment or may secrete the heterologous protein extracellularly through an outer cell wall. The strain arrays are useful for screening for improved expression of any protein of interest including therapeutic proteins, hormones, growth factors, extracellular receptors or ligands, proteases, kinases, blood proteins, chemokines, cytokines, antibodies and the like.
288 Citations
13 Claims
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1. A method for identifying an optimal population of Pseudomonas fluorescens (P. fluorescens) host cells for expression of at least one heterologous protein of interest comprising:
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a) assembling a strain array comprising at least four populations of P. fluorescens host cells, wherein each population of P. fluorescens host cells in the strain array is selected from the group consisting of; i) a population of P. fluorescens host cells that has been genetically modified to reduce the expression of at least one target gene encoding a protease, wherein the protease is an aminopeptidase, a dipeptidase, a dipeptidyl-peptidase, a tripeptidyl peptidase, a peptidyl-dipeptidase, a serine-type carboxypeptidase, a metallocarboxypeptidase, a cysteine-type carboxypeptidase, an omegapeptidase, a serine proteinase, a threonine proteinase, a cysteine proteinase, an aspartic proteinase, or a metalloproteinase; ii) a population of P. fluorescens host cells that has been genetically modified to increase the expression of at least one target gene encoding a protein that modulates protein processing, folding or translocation, wherein the protein that modulates protein processing, folding or translocation is a chaperone, a disulfide bond isomerase, a peptidyl-prolyl cis-trans isomerase, a GroES/EL folding modulator, a DnaKJ folding modulator, a Clp folding modulator, an Hsp90 folding modulator, a SecB folding modulator, a PapD chaperone, an HSP70 protein, an HSP110/SSE protein, an HSP40 (DNAJ-related) protein, a GRPE-like protein, an HSP90 protein, a CPN60 protein, a CPN10 protein, an HSP100 protein, a small HSP, a calnexin, a calreticulin, a PDI-related protein, a thioredoxin-related protein, a cyclophilin PPIase, a FK-506 binding protein, or parvulin PPIase; and
,iii) a population of P. fluorescens host cells that has been genetically modified to reduce the expression of at least one target gene encoding a protease, wherein the protease is an aminopeptidase, a dipeptidase, a dipeptidyl-peptidase, a tripeptidyl peptidase, a peptidyl-dipeptidase, a serine-type carboxypeptidase, a metallocarboxypeptidase, a cysteine-type carboxypeptidase, an omegapeptidase, a serine proteinase, a threonine proteinase, a cysteine proteinase, an aspartic proteinase, or a metalloproteinase, and to increase the expression of at least one target gene encoding a protein that modulates protein processing, folding or translocation, wherein the protein that modulates protein processing, folding or translocation is a chaperone, a disulfide bond isomerase, a peptidyl-prolyl cis-trans isomerase, a GroES/EL folding modulator, a DnaKJ folding modulator, a Clp folding modulator, an Hsp90 folding modulator, a SecB folding modulator, a PapD chaperone, an HSP70 protein, an HSP110/SSE protein, an HSP40 (DNAJ-related) protein, a GRPE-like protein, an HSP90 protein, a CPN60 protein, a CPN10 protein, an HSP100 protein, a small HSP, a calnexin, a calreticulin, a PDI-related protein, a thioredoxin-related protein, a cyclophilin PPIase, a FK-506 binding protein, or parvulin PPIase; wherein each population of P. fluorescens host cells in the strain array is non-identical and wherein each population of P. fluorescens host cells in the strain array is physically separate one from another; b) introducing into at least one cell of each of the at least four populations of P. fluorescens host cells in the strain array an expression cassette comprising at least one gene encoding the at least one heterologous protein of interest, wherein when the population of P. fluorescens host cells into which the expression cassette is introduced has been genetically modified to reduce the expression of at least one target gene encoding a protease as set forth in (i), the gene encoding the heterologous protein of interest does not encode the protease of (i), and wherein when the population of P. fluorescens host cells into which the expression cassette is introduced has been genetically modified to increase the expression of at least one target gene encoding a protein that modulates protein processing, folding or translocation as set forth in (ii), the gene encoding the heterologous protein of interest does not encode the protein that modulates protein processing, folding or translocation of (ii), and wherein when the population of P. fluorescens host cells has been genetically modified to reduce the expression of at least one target gene encoding a protease, and to increase the expression of at least one target gene encoding a protein that modulates protein processing, folding or translocation as set forth in (iii), the gene encoding the heterologous protein of interest does not encode the protease of (i) or the protein that modulates protein processing, folding or translocation of (ii); c) maintaining the populations of P. fluorescens host cells in the strain array under conditions sufficient for the expression of said heterologous protein of interest encoded by the gene encoding the at least one heterologous protein of interest, in the at least four populations of P. fluorescens host cells in the strain array, and expressing said heterologous protein of interest encoded by the gene encoding the at least one heterologous protein of interest, in the at least four populations of P. fluorescens host cells in the strain array; and
,d) selecting the optimal population of P. fluorescens host cells in the strain array in which the heterologous protein of interest encoded by the gene encoding the at least one heterologous protein of interest is produced, by simultaneous screening of the at least four populations of P. fluorescens host cells in the strain array; wherein the heterologous protein of interest encoded by the gene encoding the at least one heterologous protein of interest exhibits one or more of improved expression, improved activity, improved solubility, improved translocation, or reduced proteolytic degradation in the optimal population of P. fluorescens host cells compared to other populations of P. fluorescens host cells in the strain array. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)
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Specification