Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Enzymatic production of a protein or polypeptide
Reexamination Certificate
1998-08-10
2002-03-26
Seldha, Tekchand (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Enzymatic production of a protein or polypeptide
C435S006120, C435S069100, C435S091100, C435S233000, C435S252300, C435S320100, C536S023100, C536S023200, C530S300000, C530S350000
Reexamination Certificate
active
06361964
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to novel eukaryotic disulfide bond-forming proteins and uses thereof, particularly for increasing yields of recombinant proteins produced in in vivo or in vitro expression systems.
Many commercially produced proteins are cell surface or extracellular proteins that contain cysteine residues capable of forming disulfide bonds in the oxidizing environment of the endoplasmic reticulum (ER). For these proteins to assume their proper active folded conformation, the cysteine residues must be linked by disulfide bonds in a correct pairwise arrangement, a process that is catalyzed by cellular enzymes. One such enzyme involved in both the formation and rearrangement of disulfide bonds in eukaryotic cells is the abundant ER protein disulfide-isomerase (PDI). Protein production strategies to maximize the yield of disulfide bond-containing proteins have made use of PDI, either by overproducing PDI in cells expressing a protein of interest or by mixing a denatured protein substrate with purified PDI in in vitro refolding systems. In either case, even the use of excess PDI has generally resulted in only a modest increase in the yield of properly folded protein, and has sometimes catalyzed instead the formation of insoluble protein aggregates.
SUMMARY OF THE INVENTION
In general, the invention features a method of increasing disulfide bond formation in a protein (for example, a secreted protein) involving: (a) denaturing the protein; and (b) allowing renaturation of the protein in the presence of an Ero1 polypeptide (formerly known as a Sec81 polypeptide). In a preferred embodiment of this method, the Ero1 polypeptide is combined with a protein disulfide-isomerase. In another embodiment, the Ero1 polypeptide is derived from a yeast.
In another aspect, the invention features a method of increasing disulfide bond formation in a protein (for example, a secreted protein), involving expressing the protein in a host cell that also expresses an isolated nucleic acid that encodes an Ero1 polypeptide. In a preferred embodiment of this method, the host cell further expresses a nucleic acid encoding a protein disulfide-isomerase. In another embodiment, the Ero1 polypeptide is derived from a yeast.
In another aspect, the invention features a substantially pure preparation of an Ero1 polypeptide, which may be derived from a yeast or from a mammal (for example, a human). In preferred embodiments, the Ero1 polypeptide includes an amino acid sequence which is at least 27%, preferably at least 50%, more preferably at least 60%, and most preferably at least 80% identical to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 29, or alternatively which exhibits at least 50%, preferably, at least 70%, more preferably at least 80%, and most preferably at least 90% sequence identity to SEQ ID NOS: 3, 4, 5, 6, 7, 8, 9, or 10, or any combination thereof.
The invention also features isolated nucleic acid encoding an Ero1 polypeptide. This isolated nucleic acid is preferably at least 27%, more preferably 50%, and most preferably at least 75% identical to the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 28, or encodes an Ero1 polypeptide which either includes an amino acid sequence that is at least 27%, preferably at least 50%, more preferably at least 60%, and most preferably at least 80% identical to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 29, or exhibits at least 50%, preferably at least 70%, more preferably at least 80%, and most preferably at least 90% sequence identity to SEQ ID NOS: 3, 4, 5, 6, 7, 8, 9, or 10 or any combination thereof. This nucleic acid may include the sequence of SEQ ID NO: 1 or SEQ ID NO: 28, or, in a preferred embodiment, may complement an Ero1 mutation in yeast (for example,
S. cerevisiae
).
The isolated nucleic acid encoding an Ero1 polypeptide may be included in a vector, such as a vector that is capable of directing the expression of the protein encoded by the nucleic acid in a vector-containing cell. The isolated nucleic acid in the vector can be operatively linked to a promoter, for example, a promoter that is capable of overexpressing the Ero1 polypeptide, or that is capable of expressing Ero1 in a conditional manner. The isolated nucleic acid encoding an Ero1 polypeptide, or a vector including this nucleic acid, may be contained in a cell, such as a bacterial, mammalian, or yeast cell.
Also included in the invention is a method of producing a recombinant Ero1 polypeptide, and an Ero1 polypeptide produced by this method. This method involves (a) providing a cell transformed with isolated nucleic acid that encodes an Ero1 polypeptide and is positioned for expression in the cell under conditions for expressing the isolated nucleic acid, and (b) expressing the recombinant Ero1 polypeptide.
A substantially pure antibody, such as a monoclonal or polyclonal antibody, that specifically recognizes and binds an Ero1 polypeptide is also included in the invention. Preferably, the Ero1 polypeptide is derived from a yeast.
The invention also features a method of detecting a gene, or a portion of a gene, that is found in a mammalian cell (for example, a human cell) and that has sequence identity to the Ero1 sequence of
FIG. 1A
(SEQ ID NO: 1) or to the Ero1 sequence of
FIG. 10
(SEQ ID NO: 28). In this method, isolated nucleic acid encoding the Ero1 polypeptide, a portion of such nucleic acid greater than about 15 residues in length, or a degenerate oligonucleotide corresponding to one or more Ero1 conserved domains (for example, SEQ ID NOS: 3, 4, 5, 6, 7, 8, 9, or 10), is contacted with a preparation of nucleic acid from the mammalian (for example, human) cell under hybridization conditions that provide detection of nucleic acid sequences having about 50% or greater nucleic acid sequence identity. If desired, this method may also include a step of testing the gene, or portion thereof, for the ability to functionally complement a yeast Ero1 mutant (e.g., a
S. cerevisiae
Ero1 mutant).
Another method included in the invention is a method of isolating a gene, or a portion of a gene, that is found in a mammalian cell (for example, a human cell) and has at least 50%, preferably at least 70%, more preferably at least 80%, and most preferably at least 90% sequence identity to a sequence encoding SEQ ID NOS: 3, 4, 5, 6, 7, 8, 9, or 10. This method involves (a) amplifying by PCR the mammalian gene, or portion thereof, using oligonucleotide primers having regions of complementarity to opposite nucleic acid strands in a region of the nucleotide sequence of
FIG. 1A
(SEQ ID NO: 1) or of
FIG. 10
(SEQ ID NO: 28), and (b) isolating the mammalian gene, or portion thereof. This method can also include a step of testing the gene, or portion thereof, for the ability to functionally complement a yeast Ero1 mutant (e.g., a
S. cerevisiae
Ero1 mutant).
As used herein, by an “Ero1” polypeptide is meant a polypeptide, formerly known as a Sec81 polypeptide, derived from a eukaryote that promotes disulfide bond formation and whose function may be substituted by an exogenous oxidant, such as diamide (for example, under conditions as described herein).
By “substantially pure” is meant a preparation which is at least 60% by weight (dry weight) the compound of interest, e.g., an Ero1 polypeptide. Preferably the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99% by weight the compound of interest. Purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
By “isolated nucleic acid” is meant nucleic acid that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5′ end and one on the 3′ end) in the naturally-occurring genome of the organism from which it is derived. The term therefore includes, for example, a recombinant nucleic acid which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or e
Frand Alison R.
Kaiser Chris A.
Clark & Elbing LLP
Massachusetts Institute of Technology
Seldha Tekchand
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