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
1998-07-31
2002-06-25
Jones, W. Gary (Department: 1634)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S005000, C435S006120, C435S091100, C435S173300, C435S320100
Reexamination Certificate
active
06410264
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the area of protein production, more particularly to yeast-derived regulatory regions and coding sequences for use in the production and secretion of heterologous proteins using a yeast host expression system.
BACKGROUND OF THE INVENTION
Yeast host expression systems have successfully been used for production and secretion of heterologous proteins. Expression of a protein of interest can be enhanced with use of yeast-recognized regulatory regions. Increased yield of a heterologous protein of interest is commonly achieved with the use of yeast-derived signal and secretion leader peptide sequences. The use of native yeast secretion leaders reportedly improves direction of the protein of interest through the secretory pathway of the yeast host. Modifications to secretion leaders such as with truncation, may further improve yield.
Pichia pastoris
has proven to be a desirable yeast host for production and secretion of high levels of some heterologous proteins. Additional yeast-derived regulatory regions and native yeast secretion leaders for use in heterologous protein expression in this and other yeast hosts are needed.
SUMMARY OF THE INVENTION
Compositions and methods for expression of proteins, more particularly heterologous proteins, using a yeast host cell as the expression system are provided. Compositions of the invention are the nucleotide sequences for the promoter and terminator regions for a novel
Pichia pastoris
gene, designated PpSEC10 gene, and the nucleotide sequences and respective amino acid sequences for the secretion leader and the mature Sec10p protein components of the precursor polypeptide encoded by this novel gene.
These compositions are useful in methods for expression and secretion of proteins, particularly heterologous proteins. Vectors having at least one copy of a DNA construct comprising at least one of the PpSEC10-derived regulatory and coding nucleotide sequences in proper reading frame with a nucleotide sequence encoding a protein of interest are constructed. A yeast host cell transformed with such a vector can then be cultured and screened for secretion of the protein of interest.
A mutant
Pichia pastoris
strain that has a disabled PpSEC10 gene and which does not express the Sec10p protein is also provided for use in the methods of the present invention. The Sec10p protein is normally expressed and secreted into the culture medium at high levels. Use of the mutant yeast strain is advantageous for protein production purposes as purification of the desired protein from the culture medium is simplified.
The Sec10p protein is useful for identifying culture conditions under which the PpSEC10 promoter drives transcription of a coding sequence of interest. In this manner, antibodies to the Sec10p protein are provided for detection of this protein in the culture medium. Kits for use in the methods of protein production and detection of Sec10p protein are also provided.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to compositions and methods for expression and secretion of proteins, more particularly heterologous proteins, using a yeast host cell as the expression system. Compositions of the invention include isolated nucleotide sequences for the regulatory transcription initiation and termination regions of a novel
Pichia pastoris
gene, hereinafter designated the PpSEC10 gene, and the isolated nucleotide sequences and respective amino acid sequences for the secretion leader and for the mature Sec10p protein components of the precursor polypeptide encoded by this novel PpSEC10 gene. Variants and fragments of these PpSEC10-derived nucleotide and amino acid sequences are also encompassed by the present invention. By “isolated” is intended purified either partially or substantially as well as encompassing the use of the PpSEC10-derived nucleotide or amino acid sequences in uses other than their natural setting, for example in chimeric constructions, expression vectors, or transformation plasmids.
The PpSEC10-derived compositions disclosed herein are useful in methods directed to isolation of homologous nucleotide sequences and to expression and secretion of proteins, particularly heterologous proteins, using a yeast host expression system. These methods and additional uses for these compositions are disclosed in detail below.
The novel PpSEC10 gene of the present invention encodes a precursor polypeptide that comprises a secretion leader and a polypeptide sequence for the mature form of a 10 kDa yeast-secreted protein designated the Sec10p protein. 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 proteins: a secretion leader with a hydrophobic N-terminal sequence that is characteristic of the secretion signal, a mature protein sequence, and two basic amino acids that are positioned at the C-terminus of the secretion leader and which directly precede the mature protein 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 protein amino acid sequence is 10 kDa, while the secreted protein's estimated weight based on SDS-PAGE mobility is 18 kDa, indicating Sec10p may be glycosylated.
Wild-type
Pichia pastoris
cells secrete high levels of the mature Sec10p protein following proteolytic processing of the precursor polypeptide to remove the secretion leader that directs movement of the mature Sec10p protein through the secretory pathway of the yeast cell. As disclosed below, manipulation of the nucleotide sequence encoding the Sec10p precursor polypeptide results in a mutant strain of
Pichia pastoris
that has a disabled PpSEC10 gene and which lacks expression of the Sec10p protein. This mutant strain is useful in methods for expression and secretion of heterologous proteins in a yeast host expression system.
The regulatory transcription initiation and termination nucleotide sequences for the PpSEC10 gene, the nucleotide sequences encoding the components of the precursor polypeptide and their respective amino acid sequences, and variants and fragments of these nucleotide and amino acid sequences are of particular interest for the purposes of this invention.
A plasmid designated pKC172 and containing the cloned PpSEC10 gene was deposited with the American Type Culture Collection, Rockville, Md., on Feb. 5, 1997(accession number 98315, CMCC 4714). A plasmid ppGen2 in
E. coli
containing the cloned PpSEC10 gene (SEQ ID NO: 17) was deposited on Jun. 6, 1997 (accession number 98450, CMCC 4741). This deposit will be maintained under the terms of the Budapest Treaty. The PpSEC10 regulatory elements and coding sequences can be identified as portions of the plasmid DNA sequence set forth in SEQ ID NO: 17 as follows: the PpSEC10 promoter is set forth as nt 1180-228:7; the PpSEC10 secretion leader coding sequence is set forth as nt 2288-2443; the Sec10p mature protein coding sequences is set forth as nt 2444-2746; and the PpSEC10 transcription terminator is set forth as nt 2747-3061. These nucleotide sequences, and any amino acid sequences encoded thereby, are set forth individually in the sequence listing as SEQ ID NOS: 2-7 as identified below.
The sequence of the polynucleotides contained within the deposited materials, as well as the amino acid sequences of the polypeptides encoded thereby are incorporated herein by reference and are controlling in the event of any conflict with the written description of sequences herein.
Nucleotide sequences for the native transcription initiation region, also referred to as the promoter, and for the native transcription termination region, also referred to as the terminator, for the
Pichia pastoris
PpSEC10 gene are set forth in SEQ ID NOS: 2 and 3, respectively. By “transcription initiation and termination regions” is intended regulatory regions that flank a
Bishop Robert
Crawford Kenneth A.
Blackburn Robert P.
Chiron Corporation
Guth Joseph H.
Jones W. Gary
Spruill W. Murray
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