Iron regulation of the PpSEC10 transcriptional regulatory region of Pichia pastoris and methods of use
AssigneeChiron Corporation
InventorsRobert J. Bishop, Kenneth A. Crawford, Margaret M. Worden
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Abstract
Methods and compositions for the expression of nucleotide sequences are provided. Compositions comprise the nucleic acid sequences of the transcriptional regulatory region of the PpSEC10 gene of Pichia pastoris. Methods are provided to chemically regulate the PpSEC10 transcriptional control region by modulating the iron concentration in the culturing medium. The methods find use in regulating expression of nucleotide sequences. Furthermore, the methods of the invention can be used to regulate polypeptide expression, more particularly in regulating heterologous polypeptide expression, particularly using a yeast host cell as the expression system.
Claims54
A method of expressing a nucleotide sequence of interest said method comprising:
- stably introducing into a yeast host cell at least one DNA construct comprising an Fe-responsive transcriptional regulatory region, operably linked to a heterologous transcriptional initiation region operably linked to the nucleotide sequence of interest, and culturing said yeast host cell in media, wherein said Fe-responsive transcriptional regulatory region is a nucleotide sequence having at least 70% sequence identity to the sequence set forth in SEQ ID NO:1, wherein the sequence identity to SEQ ID NO:1 is determined using the Smith-Waterman homology search algorithm using a gap open penalty of 25 and a gap extension penalty of 5.
A method to regulate expression of a nucleotide sequence of interest said method comprising:
- stably introducing into a yeast host cell at least one DNA construct comprising an Fe-responsive transcriptional regulatory region, operably linked to a transcriptional initiation region operably linked to a nucleolide sequence of interest, and culturing said yeast host cell in media, wherein the expression of the nucleotide sequence of interest is regulated by modulating the Fe concentration of the media, said Fe-responsive transcriptional regulatory region is a nucleotide sequence having at least 70% sequence identity to the sequence set forth in SEQ ID NO:1, wherein the sequence identity to SEQ ID NO:1 is determined using the Smith-Waterman homology search algorithm using a gap open penalty of 25 and a gap extension penalty of 5.
A DNA construct comprising an Fe-responsive transcriptional regulatory region, operably linked to a heterologous transcriptional initiation region operably linked to a nucleotide sequence of interest, wherein said Fe-responsive transcriptional regulatory region is a nucleotide sequence having at least 70% sequence identity to the sequence set forth in SEQ ID NO:1, wherein the sequence identity to SEQ ID NO:1 is determined using the Smith-Waterman homology search algorithm using a gap open penalty of 25 and a gap extension penalty of 5.
A method of expressing a nucleotide sequence of interest said method comprises:
- stably introducing into a yeast host cell at least one DNA construct comprising an Fe-responsive transcriptional regulatory region operably linked to a transcriptional initiation region operably linked to said nucleotide sequence of interest, and culturing said yeast host cell in media comprising less than 1 μM Fe, wherein said Fe-responsive transcriptional regulatory region is a nucleotide sequence having at least 70% sequence identity to the sequence set forth in SEQ ID NO:1, wherein the sequence identity of SEQ ID NO:1 is determined using the Smith-Waterman homology search algorithm using a gap open penalty of 25 and a gap extension penalty of 5.
A method of expressing a nucleotide sequence of interest, said a method comprises:
- growing a yeast host cell having at least one transcriptional control region comprising a Fe-responsive transcriptional regulatory region, operably linked to said nucleotide sequence of interest, and culturing said yeast host cell in media comprising less than 1 μM Fe, wherein said Fe-responsive transcriptional regulatory region is a nucleotide sequence having at least 70% sequence identity to the sequence set forth in SEQ ID NO:1, wherein the sequence identity to SEQ ID NO:1 is determined using the Smith-Waterman homology search algorithm using a gap open penalty of 25 and a gap extention penalty of 5.
A method of regulating the expression of a nucleotide sequence of interest, said method comprises:
- growing a yeast host cell having at least one transcriptional control region comprising a Fe-responsive transcriptional regulatory region operably linked to said nucleotide sequence of interest and culturing said yeast host cell in media, wherein the Fe concentration of the culture media is modulated, said Fe-responsive transcriptional regulatory region is a nucleotide sequence having at least 70% sequence identity to the sequence set forth in SEQ ID NO:1, wherein the sequence identity to SEQ ID NO:1 is determined using the Smith-Waterman homology search algorithm using a gap open penalty of 25 and a gap extension penalty of 5.
A method of expressing a nucleotide sequence of interest said method comprising:
- stably introducing into a yeast host cell at least one DNA construct comprising an Fe-responsive transcriptional regulatory region, operably linked to a heterologous transcriptional initiation region operably linked to the nucleotide sequence of interest, and culturing said yeast host cell in media, wherein said Fe-responsive transcriptional regulatory region is selected from the group consisting of:
- a) a nucleotide sequence comprising at least 15 contiguous nucleotides of the sequence set forth in SEQ ID NO:1;
- and,
- b) a nucleotide sequence that hybridizes under stringent conditions to the complement of a nucleotide sequence set forth in SEQ ID NO:1.
A method to regulate expression of a nucleotide sequence of interest said method comprising:
- stably introducing into a yeast host cell at least one DNA construct comprising an Fe-responsive transcriptional regulatory region, operably linked to a transcriptional initiation region operably linked to a nucleotide sequence of interest, and culturing said yeast host cell in media, wherein the expression of the nucleotide sequence of interest is regulated by modulating the Fe concentration of the media, said Fe-responsive transcription regulatory region being selected from the group consisting of:
- a) a nucleotide sequence comprising at least 15 contiguous nucleotides of the sequence set forth in SEQ ID NO:1;
- and,
- b) a nucleotide sequence that hybridizes under stringent conditions to the complement of the nucleotide sequence comprising the sequence set forth in SEQ ID NO:1.
A DNA construct comprising an Fe-responsive transcriptional regulatory region, operably linked to a heterologous transcriptional initiation region operably linked to a nucleotide sequence of interest, wherein said Fe-responsive transcriptional regulatory region is selected from the group consisting of:
- a) a nucleotide sequence that hybridizes under stringent conditions to the complement of a nucleotide sequence comprising at least 15 contiguous nucleotides of the sequence set forth in SEQ ID NO:1;
- and,
- b) a nucleotide sequence that hybridizes under stringent conditions to the complement of a nucleotide sequence having at least 70% sequence identity to the sequence set forth in SEQ ID NO:1, wherein the sequence identity to SEQ ID NO:1 is determined using the Smith-Waterman homology search algorithm using a gap open penalty of 25 and a gap extension penalty of 5.
A method of expressing a nucleotide sequence of interest said method comprises:
- stably introducing into a yeast host cell at least one DNA construct comprising a Fe-responsive transcriptional regulatory region operably linked to a transcriptional initiation region operably linked to said nucleotide sequence of interest, and culturing said yeast host cell in media comprising less than 1 μM Fe, wherein said Fe-responsive transcriptional regulatory region is selected from the group consisting of:
- a) a nucleotide sequence that hybridizes under stringent conditions to the complement of a nucleotide sequence comprising the sequence set forth in SEQ ID NO:1;
- and,
- b) a nucleotide sequence comprising at least 15 contiguous nucleotides of the sequence set forth in SEQ ID NO:1.
A method of expressing a nucleotide sequence of interest, said method comprises:
- growing a yeast host cell having at least one transcription control region comprising a Fe-responsive transcriptional regulatory region operably linked to said nucleotide sequence of interest, and culturing said yeast host cell in media comprising less than 1 μM Fe, wherein said Fe-responsive transcriptional regulatory region is selected from the group consisting of:
- a) a nucleotide sequence that hybridizes under stringent conditions to the complement of a nucleotide sequence comprising the sequences set forth in SEQ ID NO:1;
- and,
- b) a nucleotide sequence comprising at least 15 contiguous nucleotides of the sequence set forth in SEQ ID NO:1.
A method of regulating the expression of a nucleotide sequence of interest, said method comprises:
- growing a yeast host cell having at least one transcriptional control region comprising a Fe-responsive transcriptional regulatory region operably linked to said nucleotide sequence of interest and culturing said yeast host cell in media, wherein the Fe concentration of the culture media is modulated, said Fe-responsive transcriptional regulatory region being selected from the group consisting of:
- a) a nucleotide sequence that hybridizes under stringent conditions to the complement of a nucleotide sequence comprising the sequence set forth in SEQ ID NO:1;
- and,
- b) a nucleotide sequence comprising at least 15 contiguous nucleotides of the sequence set forth in SEQ ID NO:1.
Dependencies are inferred from the claim text.