Expression of processed recombinant lactoferrin and lactoferrin polypeptide fragments from a fusion product in Aspergillus
First Claim
1. A plasmid DNA sequence comprising nucleotide sequence elements operably linked in the following 5'"'"' to 3'"'"' order:
- (a) a fungal promoter sequence;
(b) a sequence encoding a signal peptide;
(c) a sequence encoding an amino terminal portion of a highly expressed endogenous gene the product of which is secreted by Aspergillus cells; and
,(d) a sequence selected from the group consisting of a nucleotide sequence encoding a mature, native, lactoferrin, a nucleotide sequence encoding an iron-binding lobe of a native lactoferrin and a nucleotide sequence encoding an antimicrobial peptide of a native lactoferrin.
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Accused Products
Abstract
The subject invention provides for the production of lactoferrins and lactoferrin polypeptide fragments using the host cells Aspergillus in combination with novel plasmid constructs. More specifically, the subject invention provides novel vector constructs capable of producing lactoferrins and lactoferrin polypeptide fragments in Aspergillus host cells. More particularly, the subject invention provides for novel plasmid constructs suitable for use with Aspergillus and especially Aspergillus awamori, niger and oryzae host cells, which enables them to produce large amounts of recombinant lactoferrins and lactoferrin polypeptide fragments.
45 Citations
74 Claims
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1. A plasmid DNA sequence comprising nucleotide sequence elements operably linked in the following 5'"'"' to 3'"'"' order:
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(a) a fungal promoter sequence; (b) a sequence encoding a signal peptide; (c) a sequence encoding an amino terminal portion of a highly expressed endogenous gene the product of which is secreted by Aspergillus cells; and
,(d) a sequence selected from the group consisting of a nucleotide sequence encoding a mature, native, lactoferrin, a nucleotide sequence encoding an iron-binding lobe of a native lactoferrin and a nucleotide sequence encoding an antimicrobial peptide of a native lactoferrin. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 59, 60, 61, 62)
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29. A plasmid DNA sequence comprising the following components operably linked from 5'"'"' to 3'"'"':
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(a) a promoter from A. niger glucoamylase gene; (b) a signal peptide-encoding sequence from the A. niger glucoamylase gene; (c) a sequence encoding an amino terminal portion of the A. niger glucoamylase gene; (d) a sequence encoding a peptide linker comprising a Kex2 peptidase cleavage site whereby there is an endogenous proteolytic enzyme specific for said linker sequence; (e) a nucleotide sequence selected from the group consisting of a nucleotide sequence encoding a mature, native, lactoferrin, a nucleotide sequence encoding an iron-binding lobe of a native lactoferrin and a nucleotide sequence encoding an antimicrobial peptide of a native lactoferrin; (f) a transcription termination sequence from the A. niger glucoamylase gene; and (g) a phleomycin resistance selectable marker gene.
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30. A plasmid DNA sequence comprising the following components operably linked from 5'"'"' to 3'"'"':
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(a) a promoter from A. oryzae α
-amylase gene;(b) a signal peptide-encoding sequence from the A. oryzae α
-amylase gene;(c) a sequence encoding an amino terminal portion of the A. oryzae α
-amylase gene;(d) a sequence encoding a peptide linker comprising a Kex2 peptidase cleavage site whereby there is an endogenous proteolytic enzyme specific for said linker sequence; (e) a nucleotide sequence selected from the group consisting of a nucleotide sequence encoding a mature, native, lactoferrin, a nucleotide sequence encoding an iron-binding lobe of a native lactoferrin and a nucleotide sequence encoding an antimicrobial peptide of a native lactoferrin; (f) a transcription termination sequence from the A. niger glucoamylase gene; and (g) a phleomycin resistance selectable marker gene. - View Dependent Claims (65, 66)
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31. A plasmid DNA sequence comprising the following components operably linked from 5'"'"' to 3'"'"':
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(a) a promoter from A. awamori glucoamylase gene; (b) a signal peptide-encoding sequence from the A. awamori glucoamylase gene; (c) a sequence encoding an amino terminal portion of the A. awamori glucoamylase gene; (d) a sequence encoding a peptide linker comprising a Kex2 peptidase cleavage site whereby there is an endogenous proteolytic enzyme specific for said linker sequence; (e) a nucleotide sequence selected from the group consisting of a nucleotide sequence encoding a mature, native, lactoferrin, a nucleotide sequence encoding an iron-binding lobe of a native lactoferrin and a nucleotide sequence encoding an antimicrobial peptide of a native lactoferrin; (f) a transcription termination sequence from the A. niger glucoamylase gene; and (g) a phleomycin resistance selectable marker gene. - View Dependent Claims (32, 33, 63, 64)
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34. A plasmid DNA sequence comprising nucleotide sequence elements operably linked in the following 5'"'"' to 3'"'"' order:
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(a) a first DNA sequence as a means for encoding a fungal promoter sequence; (b) a second DNA sequence as a means for encoding a signal peptide; (c) a third DNA sequence as a means for encoding an amino terminal portion of a highly expressed endogenous gene the product of which is secreted by Aspergillus cells; and
,(d) a fourth DNA sequence as a means for encoding a peptide selected from the group consisting of a mature, native, lactoferrin, an iron-binding lobe of a native lactoferrin, and an antimicrobial peptide of a native lactoferrin. - View Dependent Claims (35)
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36. A process for producing lactoferrin which comprises culturing a transformed Aspergillus fungal cell containing a recombinant plasmid, wherein said plasmid comprises the following components operably linked from 5'"'"' to 3'"'"':
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(a) a promoter; (b) a signal sequence; (c) a sequence encoding an amino terminal portion of a highly expressed endogenous gene the product of which is secreted by Aspergillus cells; and
,(d) a nucleotide sequence selected from the group consisting of a nucleotide sequence encoding a mature, native, lactoferrin, a nucleotide sequence encoding an iron-binding lobe of a native lactoferrin and a nucleotide sequence encoding an antimicrobial peptide of a native lactoferrin; wherein said transformed Aspergillus fungal cells are cultured in a suitable nutrient medium until the mature, native, lactoferrin, the iron-binding lobe of a native lactoferrin, or the antimicrobial peptide of a native lactoferrin, is produced as a fusion product and then processed via an endogenous proteolytic enzyme specific for the linker sequence, wherein the processed mature, native, lactoferrin, the iron-binding lobe of a native lactoferrin, or the antimicrobial peptide of a native lactoferrin is secreted into the nutrient medium and isolated therefrom. - View Dependent Claims (37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 67, 68, 69, 70, 71, 72)
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55. A process for producing a mature, native, lactoferrin, an iron-binding lobe of a native lactoferrin, or an antimicrobial peptide of a native lactoferrin which comprises culturing a transformed Aspergillas niger fungal cell containing a recombinant plasmid, wherein said plasmid comprises the following components operably linked from 5'"'"' to 3'"'"':
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(a) a promoter from A. niger glucoamylase gene; (b) a signal peptide-encoding sequence from the A. niger glucoamylase gene; (c) a sequence encoding an amino terminal of the A. niger glucoamylase gene; (d) a sequence encoding a peptide linker comprising a Kex2 peptidase cleavage site whereby there is an endogenous proteolytic enzyme specific for said linker sequence; (e) a nucleotide sequence selected from the group consisting of a nucleotide sequence encoding a mature, native, lactoferrin, a nucleotide sequence encoding an iron-binding lobe of a native lactoferrin and a nucleotide sequence encoding an antimicrobial peptide of a native lactoferrin; (f) a transcription termination sequence from the A. niger glucoamylase gene; and (g) a phleomycin resistance selectable marker gene;
wherein said transformed Aspergillus niger fungal cell is cultured in a suitable nutrient medium until the mature, native, lactoferrin, the iron-binding lobe of a native lactoferrin, or the antimicrobial peptide of a native lactoferrin is produced as a fusion product and then processed via an endogenous proteolytic enzyme specific for said linker sequence, wherein the processed mature, native, lactoferrin, the processed iron-binding lobe of a native lactoferrin, or the processed antimicrobial peptide of a native lactoferrin is secreted into the nutrient medium and isolated therefrom.
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56. A process for producing lactoferrin which comprises culturing a transformed Aspergillus oryzae fungal cell containing a recombinant plasmid, wherein said plasmid comprises the following components operably linked from 5'"'"' to 3'"'"':
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(a) a promoter from A. oryzae α
-amylase gene;(b) a signal peptide-encoding sequence from the A. oryzae α
-amylase gene;(c) a sequence encoding an amino terminal portion of the A. oryzae α
-amylase gene;(d) a sequence encoding a peptide linker comprising a Kex2 peptidase cleavage site whereby there is an endogenous proteolytic enzyme specific for said linker sequence; (e) a nucleotide sequence selected from the group consisting of a nucleotide sequence encoding a mature, native, lactoferrin, a nucleotide sequence encoding an iron-binding lobe of a native lactoferrin and a nucleotide sequence encoding an antimicrobial peptide of a native lactoferrin; (f) a transcription termination sequence from the A. niger glucoamylase gene; and (g) a phleomycin resistance selectable marker gene;
wherein said transformed Aspergillus oryzae fungal cell is cultured in a suitable nutrient medium until the mature, native, lactoferrin, the iron-binding lobe of a native lactoferrin, or the antimicrobial peptide of a native lactoferrin is produced as a fusion product and then processed via an endogenous proteolytic enzyme specific for said linker sequence, wherein the processed mature, native, lactoferrin, the processed iron-binding lobe of a native lactoferrin, or the processed antimicrobial peptide of a native lactoferrin is secreted into the nutrient medium and isolated therefrom.
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57. A process for producing lactoferrin which comprises culturing a transformed Aspergillus awamori fungal cell containing a recombinant plasmid, wherein said plasmid comprises the following components operably linked from 5'"'"' to 3'"'"':
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(a) a promoter from A. awamori glucoamylase gene; (b) a signal peptide-encoding sequence from the A. awamori glucoamylase gene; (c) a sequence encoding an amino terminal portion of the A. awamori glucoamylase gene; (d) a sequence encoding a peptide linker comprising a Kex2 peptidase cleavage site whereby there is an endogenous proteolytic enzyme specific for said linker sequence; (e) a nucleotide sequence selected from the group consisting of a nucleotide sequence encoding a mature, native, human lactoferrin, a nucleotide sequence encoding an iron-binding lobe of a native human lactoferrin and a nucleotide sequence encoding an antimicrobial peptide of a native human lactoferrin; (f) a transcription termination sequence from the A. niger glucoamylase gene; and (g) a phleomycin resistance selectable marker gene;
wherein said transformed Aspergillus awamori fungal cell is cultured in a suitable nutrient medium until the human lactoferrin, the iron-binding lobe of a native human lactoferrin or the antimicrobial peptide of a native human lactoferrin is produced as a fusion product and then processed via an endogenous proteolytic enzyme specific for said linker sequence, wherein the processed mature, native, human lactoferrin, the processed iron-binding lobe of native human lactoferrin, or the processed antimicrobial peptide of a native human lactoferrin is secreted into the nutrient medium and isolated therefrom.
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58. A method of isolating a mature, native, lactoferrin, an iron-binding lobe of a native lactoferrin, or an antimicrobial peptide of a native lactoferrin from fungal nutrient medium comprising culturing a transformed Aspergillus awamori fungal cell containing a recombinant plasmid vector, wherein said plasmid vector comprises a promoter from A. awamori glucoamylase gene, a signal peptide-encoding sequence from the A. awamori glucoamylase gene, a sequence encoding an amino terminal of the A. awamori glucoamylase gene, a sequence encoding a peptide linker comprising a Kex2 peptidase cleavage site whereby there is an endogenous proteolytic enzyme is specific for said linker sequence, a nucleotide sequence selected from the group consisting of a nucleotide sequence encoding a mature, native, lactoferrin, a nucleotide sequence encoding an iron-binding lobe of a native lactoferrin and a nucleotide sequence encoding an antimicrobial peptide of a native lactoferrin, a transcription termination sequence from the A. niger glucoamylase gene, and a phleomycin resistance selectable marker gene and wherein said transformed Aspergillus awamori fungal cells are cultured in a suitable nutrient medium until the mature, native, lactoferrin, the iron-binding lobe of a native lactoferrin, or the antimicrobial peptide of a native lactoferrin is produced as a fusion product and then processed via an endogenous proteolytic enzyme specific for said linker sequence, wherein the processed mature, native, lactoferrin, the processed iron-binding lobe of a native lactoferrin, or the processed antimicrobial peptide of a native lactoferrin is secreted into the nutrient medium and isolated therefrom.
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73. A mature, native, lactoferrin, an iron-binding lobe of a native lactoferrin, or an antimicrobial peptide of native lactoferrin produced by a process which comprises culturing a transformed Aspergillas awamori fungal cell containing a recombinant plasmid, wherein said plasmid comprises the following components operably linked from 5'"'"' to 3'"'"':
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(a) a promoter from A. awamori glucoamylase gene; (b) a signal peptide-encoding sequence from the A. awamori glucoamylase gene; (c) a sequence encoding an amino terminal portion of the A. awamori glucoamylase gene; (d) a sequence encoding a peptide linker comprising a Kex2 peptidase cleavage site whereby there is an endogenous proteolytic enzyme specific for said linker sequence; (e) a nucleotide sequence selected from the group consisting of a nucleotide sequence encoding a mature, native, lactoferrin, a nucleotide sequence encoding an iron-binding lobe of a native lactoferrin and a nucleotide sequence encoding an antimicrobial peptide of a native lactoferrin; (f) a transcription termination sequence from the A. niger glucoamylase gene; and (g) a phleomycin resistance selectable marker gene; wherein said transformed Aspergillus awamori fungal cell is cultured in a suitable nutrient medium until the mature, native, lactoferrin, the iron-binding lobe of a native lactoferrin, or the antimicrobial peptide of a native lactoferrin is produced as a fusion product and then processed via an endogenous proteolytic enzyme specific for said linker sequence, wherein the processed mature, native, lactoferrin, the processed iron-binding lobe of a native lactoferrin, or the processed antimicrobial peptide of a native lactoferrin is secreted into the nutrient medium and isolated therefrom. - View Dependent Claims (74)
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Specification