Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2001-05-24
2003-08-12
McKelvey, Terry (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S320100, C435S483000, C536S024100
Reexamination Certificate
active
06605450
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the expression of nucleic acid sequences of interest.
BACKGROUND OF THE INVENTION
The PpSEC10 gene of
P. pastoris
encodes a precursor polypeptide that comprises a secretion leader and a polypeptide sequence for the mature form of a 10 kDa yeast-secreted polypeptide designated the SEC10p polypeptide. This precursor polypeptide represents the initial translation product of mRNA transcribed from the PpSEC10 gene. The PpSEC10 precursor polypeptide has some structural components that are typical of secreted polypeptides: a secretion leader with a hydrophobic N-terminal sequence that is characteristic of the secretion signal, a mature polypeptide sequence, and two basic amino acids that are positioned at the C-terminus of the secretion leader and which directly precede the mature polypeptide sequence. Dibasic residues are a common cleavage recognition sequence for processing proteases such as Kex2.
The predicted molecular weight of the mature form of SEC10p based on the amino acid sequence is 10 kDa, while the estimated molecular weight of the secreted polypeptide based on SDS-PAGE mobility is 18 kDa, indicating SEC10p is glycosylated. Wild-type
Pichia pastoris
cells secrete high levels of the mature SEC10p polypeptide following proteolytic processing of the precursor polypeptide to remove the secretion leader that directs movement of the mature SEC10p polypeptide through the secretory pathway of the yeast cell.
The regulatory and coding sequences of the PpSEC10 gene are provided in Publication No. WO 99/24062, entitled “Novel
Pichia pastoris
Gene Sequences and Methods for Their Use,” herein incorporated by reference. Nucleotide sequences comprising the PpSEC10 gene were deposited with the American Type Culture Collection, Rockville, Md., on Feb. 5, 1997 (Accession No. 98315) and on Jun. 6, 1997 (Accession No. 98450).
The present invention provides methods and compositions for the regulated expression of nucleic acid sequences using the transcriptional regulatory region of the PpSEC10 gene of
Pichia pastoris.
The methods find use in the expression of nucleotide sequences of interest and in the production of commercially significant concentrations of heterologous polypeptides.
SUMMARY OF THE INVENTION
Methods and compositions are provided for the expression of a nucleotide sequence of interest using regulatory sequences of the PpSEC10 gene of
Pichia pastoris.
Methods comprise means to chemically regulate the PpSEC10 transcriptional control region by modulating 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.
Compositions of the invention include a DNA construct comprising a transcriptional regulatory region, operably linked to a heterologous transcriptional initiation region operably linked to a nucleotide sequence of interest, wherein said transcriptional regulatory region comprises the sequence set forth in SEQ ID NO:1 or a variant or fragment thereof. Compositions of the invention further include an expression vector comprising this DNA construct.
Methods are provided to regulate the expression of a nucleotide sequence of interest comprising stably introducing into the genome of a yeast host cell at least one DNA construct comprising, in proper reading frame, an Fe-responsive PpSEC10 transcriptional regulatory region comprising the nucleotide sequence set forth in SEQ ID NO:1 or a variant or fragment thereof, a heterologous transcriptional initiation region, and a nucleotide sequence encoding said nucleotide sequence of interest, and culturing said yeast cells in media.
Methods of the invention also provide a means to regulate expression of a nucleotide sequence of interest comprising, stably introducing into a yeast host at least one DNA construct comprising, in proper reading frame an Fe-responsive transcriptional regulatory region from PpSEC10 comprising the sequence set forth in SEQ ID NO:1 or a fragment or variant thereof, a transcriptional initiation region, and a nucleotide sequence of interest, culturing said yeast host cell to generate cell mass, wherein the Fe concentration is sufficient to at least partially repress the expression of the nucleotide sequence of interest, reducing the concentration of Fe in the media to induce increased expression of the nucleotide sequence of interest, and further culturing the yeast host until a desired amount of the nucleotide sequence of interest or the polypeptide encoded by the DNA sequence is produced.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to methods and compositions for the expression of a nucleotide sequence of interest. The methods of the invention can also be used for the expression and isolation of polypeptides, for example, heterologous polypeptides, using a yeast host cell as the expression system. Compositions comprise isolated or purified nucleic acid sequences that comprise the nucleic acid sequences of the transcriptional regulatory element of the PpSEC10 gene (SEQ ID NO:1) and fragments and variants thereof.
The invention encompasses isolated or substantially purified nucleic acid or protein compositions. A “purified” nucleic acid molecule or protein, or biologically active portion thereof, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. A polypeptide that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, 5%, (by dry weight) of contaminating protein. When the polypeptide of the invention or biologically active portion thereof is recombinantly produced, preferably culture medium represents less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or non-protein-of-interest chemicals. An “isolated” nucleic acid is free of sequences (preferably protein encoding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.
The present invention provides methods to chemically regulate the PpSEC10 transcriptional control region by modulating the Fe concentration of the culturing medium. Hence the methods of the invention find use in regulating expression of a nucleotide sequence of interest. By “transcriptional control region” is intended the nucleotide sequences that control transcription. The transcriptional control region contains at least two domains. The first domain is a transcriptional initiation region, which includes the transcription initiation site, the TATA box, RNA capping sequences as appropriate, and an RNA polymerase binding site. The second domain of the transcriptional control region is the transcriptional regulatory region or upstream activating sequence (UAS). The transcriptional regulatory region may regulate transcription in a positive or negative manner resulting in either the enhancement or repression of transcription, respectively.
A transcriptional regulatory region (SEQ ID NO:1) within the transcriptional control region (SEQ ID NO:2) of the PpSEC10 nucleotide sequence has been identified by the inventors. The PpSEC10 regulatory region is located upstream of the transcription initiation site and is responsible for the Fe regulation of the PpSEC10 transcriptional initiation region. Methods to regulate the PpSEC10 transcriptional initiation region using the Fe-responsiv
Bishop Robert J.
Crawford Kenneth A.
Worden Margaret M.
Blackburn Robert P.
Chiron Corporation
Guth Joseph H.
McKelvey Terry
Spruill W. Murray
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