# US 5,700,678 A · Protein disulfide-isomerase and production thereof

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- Number: US 5,700,678 A
- Document ID: US5700678A
- Status: Granted
- Kind: A
- Application number: 07/982,138
- Filed: 1992-11-25
- Granted: 1997-12-23
- Earliest claimed: 1987-06-01
- Assignees: Takeda Chemical Industries, Ltd.
- Inventors: Kumao Toyoshima; Ryuya Horiuchi; Kiyoshi Yamauchi; Tadashi Yamamoto; Koichi Igarashi
- Web page: https://patentgrep.com/patent/US5700678A

## CPC

- C07K 1/1133: {by redox-reactions involving cystein/cystin side chains}
- C07K 14/55: IL-2
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/90: Isomerases (5.)
- C12Y 503/04001: Protein disulfide-isomerase (5.3.4.1), i.e. disufide bond-forming enzyme
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES

## Abstract

Protein disulfide isomerase, which catalyzes the exchange reaction between sulfhydryl and disulfide in a protein for formation of the most stable natural type disulfide bonds, is useful for formation of natural type disulfide bonds in a protein which is produced in a prokaryotic cell.

## Claims

### Claim 1

An isolated DNA encoding the amino acid sequence of FIG. 3.

### Claim 2

An isolated DNA encoding the amino acid sequence:
______________________________________ MetAlaProGluGluGluAspHisValLeuValLeuArgLysSerAsnPheAlaGluAla LeuAlaAlaHisLysTyrLeuLeuValGluPheTyrAlaProTrpCysGlyHisCysLys AlaLeuAlaProGluTyrAlaLysAlaAlaGlyLysLeuLysAlaGluGlySerGluIle ArgLeuAlaLysValAspAlaThrGluGluSerAspLeuAlaGlnGlnTyrGlyValArg GlyTyrProThrIleLysPhePheArgAsnGlyAspThrAlaSerProLysGluTyrThr AlaGlyArgGluAlaAspAspIleValAsnTrpLeuLysLysArgThrGlyProAlaAla ThrThrLeuProAspGlyAlaAlaAlaGluSerLeuValGluSerSerGluValAlaVal IleGlyPhePheLysAspValGluSerAspSerAlaLysGlnPheLeuGlnAlaAlaGlu AlaIleAspAspIleProPheGlyIleThrSerAsnSerAspValPheSerLysTyrGln LeuAspLysAspGlyValValLeuPheLysLysPheAspGluGlyArgAsnAsnPheGlu GlyGluValThrLysGluAsnLeuLeuAspPheIleLysHisAsnGlnLeuProLeuVal IleGluPheThrGluGlnThrAlaProLysIlePheGlyGlyGluIleLysThrHisIle LeuLeuPheLeuProLysSerValSerAspTyrAspGlyLysLeuSerAsnPheLysThr AlaAlaGluSerPheLysGlyLysIleLeuPheIlePheIleAspSerAspHisThrAsp AsnGlnArgIleLeuGluPhePheGlyLeuLysLysGluGluCysProAlaValArgLeu IleThrLeuGluGluGluMetThrLysTyrLysProGluSerGluGluLeuThrAlaGlu ArgIleThrGluPheCysHisArgPheLeuGluGlyLysIleLysProHisLeuMetSer GlnGluLeuProGluAspTrpAspLysGlnProValLysValLeuValGlyLysAsnPhe GluAspValAlaPheAspGluLysLysAsnValPheValGluPheTyrAlaProTrpCys GlyHisCysLysGlnLeuAlaProIleTrpAspLysLeuGlyGluThrTyrLysAspHis GluAsnIleValIleAlaLysMetAspSerThrAlaAsnGluValGluAlaValLysVal HisSerPheProThrLeuLysPhePheProAlaSerAlaAspArgThrValIleAspTyr AsnGlyGluArgThrLeuAspGlyPheLysLysPheLeuGluSerGlyGlyGlnAspGly AlaGlyAspAspAspAspLeuGluAspLeuGluGluAlaGluGluProAspMetGluGlu AspAspAspGlnLysAlaValLysAspGluLeu. ______________________________________

### Claim 3 (depends on 2)

The isolated DNA of claim 2, wherein said isolated DNA comprises the nucleotide sequence of nucleotide 104 to nucleotide 1573 of FIG. 5.

### Claim 4

A recombinant vector comprising a nucleotide sequence which encodes a polypeptide comprising the amino acid sequence of FIG. 3 and which is operably linked to a promoter sequence, wherein said recombinant vector replicates and expresses said polypeptide when transformed into in a microorganism.

### Claim 5 (depends on 4)

The recombinant vector of claim 4, wherein said microorganism is Escherichia coli.

### Claim 6 (depends on 4)

The recombinant vector of claim 4 which is recombinant vector pTB766 carried by Escherichia coli MM294/pTB766 (IFO 14611 FERM BP-1842) or is recombinant vector pTB767 carried by Saccharomyces cerevisiae AH22R.sup.- /pTB767 (IFO 10425, FERM BP-1843).

### Claim 7 (depends on 4)

The recombinant vector of claim 4, wherein said nucleotide sequence encodes the following amino acid sequence:
______________________________________ MetAlaProGluGluGluAspHisValLeuValLeuArgLysSerAsnPheAlaGluAla LeuAlaAlaHisLysTyrLeuLeuValGluPheTyrAlaProTrpCysGlyHisCysLys AlaLeuAlaProGluTyrAlaLysAlaAlaGlyLysLeuLysAlaGluGlySerGluIle ArgLeuAlaLysValAspAlaThrGluGluSerAspLeuAlaGlnGlnTyrGlyValArg GlyTyrProThrIleLysPhePheArgAsnGlyAspThrAlaSerProLysGluTyrThr AlaGlyArgGluAlaAspAspIleValAsnTrpLeuLysLysArgThrGlyProAlaAla ThrThrLeuProAspGlyAlaAlaAlaGluSerLeuValGluSerSerGluValAlaVal IleGlyPhePheLysAspValGluSerAspSerAlaLysGlnPheLeuGlnAlaAlaGlu AlaIleAspAspIleProPheGlyIleThrSerAsnSerAspValPheSerLysTyrGln LeuAspLysAspGlyValValLeuPheLysLysPheAspGluGlyArgAsnAsnPheGlu GlyGluValThrLysGluAsnLeuLeuAspPheIleLysHisAsnGlnLeuProLeuVal IleGluPheThrGluGlnThrAlaProLysIlePheGlyGlyGluIleLysThrHisIle LeuLeuPheLeuProLysSerValSerAspTyrAspGlyLysLeuSerAsnPheLysThr AlaAlaGluSerPheLysGlyLysIleLeuPheIlePheIleAspSerAspHisThrAsp AsnGlnArgIleLeuGluPhePheGlyLeuLysLysGluGluCysProAlaValArgLeu IleThrLeuGluGluGluMetThrLysTyrLysProGluSerGluGluLeuThrAlaGlu ArgIleThrGluPheCysHisArgPheLeuGluGlyLysIleLysProHisLeuMetSer GlnGluLeuProGluAspTrpAspLysGlnProValLysValLeuValGlyLysAsnPhe GluAspValAlaPheAspGluLysLysAsnValPheValGluPheTyrAlaProTrpCys GlyHisCysLysGlnLeuAlaProIleTrpAspLysLeuGlyGluThrTyrLysAspHis GluAsnIleValIleAlaLysMetAspSerThrAlaAsnGluValGluAlaValLysVal HisSerPheProThrLeuLysPhePheProAlaSerAlaAspArgThrValIleAspTyr AsnGlyGluArgThrLeuAspGlyPheLysLysPheLeuGluSerGlyGlyGlnAspGly AlaGlyAspAspAspAspLeuGluAspLeuGluGluAlaGluGluProAspMetGluGlu AspAspAspGlnLysAlaValLysAspGluLeu. ______________________________________

### Claim 8

transformed microorganism comprising a recombinant vector, wherein said recombinant vector comprises a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of FIG. 3, which nucleotide sequence is operably linked to a promoter sequence, and wherein said recombinant vector replicates and expresses said polypeptide in said transformed microorganism.

### Claim 9 (depends on 8)

The transformed microorganism of claim 8, wherein said nucleotide sequence comprises nucleotide 104 to nucleotide 1573 of FIG. 5.

### Claim 10 (depends on 8)

The transformed microorganism of claim 8, wherein said transformed microorganism is Escherichia coli.

### Claim 11 (depends on 8)

The transformed microorganism of claim 8 which is Escherichia coli MM294/pTB766 (IFO 14611 FERM BP-1842) or Saccharomyces cerevisiae AH22R.sup.- /pTB 767 (IFO 10425, FERM BP-1843).

### Claim 12 (depends on 8)

The transformed microorganism of claim 8, wherein said nucleotide sequence encodes the following amino acid sequence:
______________________________________ MetAlaProGluGluGluAspHisValLeuValLeuArgLysSerAsnPheAlaGluAla LeuAlaAlaHisLysTyrLeuLeuValGluPheTyrAlaProTrpCysGlyHisCysLys AlaLeuAlaProGluTyrAlaLysAlaAlaGlyLysLeuLysAlaGluGlySerGluIle ArgLeuAlaLysValAspAlaThrGluGluSerAspLeuAlaGlnGlnTyrGlyValArg GlyTyrProThrIleLysPhePheArgAsnGlyAspThrAlaSerProLysGluTyrThr AlaGlyArgGluAlaAspAspIleValAsnTrpLeuLysLysArgThrGlyProAlaAla ThrThrLeuProAspGlyAlaAlaAlaGluSerLeuValGluSerSerGluValAlaVal IleGlyPhePheLysAspValGluSerAspSerAlaLysGlnPheLeuGlnAlaAlaGlu AlaIleAspAspIleProPheGlyIleThrSerAsnSerAspValPheSerLysTyrGln LeuAspLysAspGlyValValLeuPheLysLysPheAspGluGlyArgAsnAsnPheGlu GlyGluValThrLysGluAsnLeuLeuAspPheIleLysHisAsnGlnLeuProLeuVal IleGluPheThrGluGlnThrAlaProLysIlePheGlyGlyGluIleLysThrHisIle LeuLeuPheLeuProLysSerValSerAspTyrAspGlyLysLeuSerAsnPheLysThr AlaAlaGluSerPheLysGlyLysIleLeuPheIlePheIleAspSerAspHisThrAsp AsnGlnArgIleLeuGluPhePheGlyLeuLysLysGluGluCysProAlaValArgLeu IleThrLeuGluGluGluMetThrLysTyrLysProGluSerGluGluLeuThrAlaGlu ArgIleThrGluPheCysHisArgPheLeuGluGlyLysIleLysProHisLeuMetSer GlnGluLeuProGluAspTrpAspLysGlnProValLysValLeuValGlyLysAsnPhe GluAspValAlaPheAspGluLysLysAsnValPheValGluPheTyrAlaProTrpCys GlyHisCysLysGlnLeuAlaProIleTrpAspLysLeuGlyGluThrTyrLysAspHis GluAsnIleValIleAlaLysMetAspSerThrAlaAsnGluValGluAlaValLysVal HisSerPheProThrLeuLysPhePheProAlaSerAlaAspArgThrValIleAspTyr AsnGlyGluArgThrLeuAspGlyPheLysLysPheLeuGluSerGlyGlyGlnAspGly AlaGlyAspAspAspAspLeuGluAspLeuGluGluAlaGluGluProAspMetGluGlu AspAspAspGlnLysAlaValLysAspGluLeu. ______________________________________

### Claim 13

A method for producing a polypeptide comprising the amino acid sequence of FIG. 3, which method comprises the steps of: (i) cultivating a transformed microorganism comprising a recombinant vector, wherein said recombinant vector comprises a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of FIG. 3, which nucleotide sequence is operably linked to a promoter sequence, and wherein said recombinant vector replicates and expresses said polypeptide in said transformed microorganism; (ii) producing and accumulating said polypeptide intracellularly in said transformed microorganism; and (iii) recovering said polypeptide from said transformed microorganism.

### Claim 14 (depends on 13)

method of claim 13, wherein said nucleotide sequence comprises nucleotide 104 to nucleotide 1573 of FIG. 5.

### Claim 15 (depends on 13)

The method of claim 13, wherein said transformed microorganism is Escherichia coli.

### Claim 16 (depends on 13)

The method of claim 13, wherein said microorganism is Escherichia coli MM294/pTB766 (IFO 14611 FERM BP-1842) or Saccharomyces cerevisiae AH22R.sup.- /pTB767 (IFO 10425, FERM BP-1843).

### Claim 17 (depends on 13)

The method of claim 13, wherein said nucleotide sequence encodes the following amino acid sequence:
______________________________________ MetAlaProGluGluGluAspHisValLeuValLeuArgLysSerAsnPheAlaGluAla LeuAlaAlaHisLysTyrLeuLeuValGluPheTyrAlaProTrpCysGlyHisCysLys AlaLeuAlaProGluTyrAlaLysAlaAlaGlyLysLeuLysAlaGluGlySerGluIle ArgLeuAlaLysValAspAlaThrGluGluSerAspLeuAlaGlnGlnTyrGlyValArg GlyTyrProThrIleLysPhePheArgAsnGlyAspThrAlaSerProLysGluTyrThr AlaGlyArgGluAlaAspAspIleValAsnTrpLeuLysLysArgThrGlyProAlaAla ThrThrLeuProAspGlyAlaAlaAlaGluSerLeuValGluSerSerGluValAlaVal IleGlyPhePheLysAspValGluSerAspSerAlaLysGlnPheLeuGlnAlaAlaGlu AlaIleAspAspIleProPheGlyIleThrSerAsnSerAspValPheSerLysTyrGln LeuAspLysAspGlyValValLeuPheLysLysPheAspGluGlyArgAsnAsnPheGlu GlyGluValThrLysGluAsnLeuLeuAspPheIleLysHisAsnGlnLeuProLeuVal IleGluPheThrGluGlnThrAlaProLysIlePheGlyGlyGluIleLysThrHisIle LeuLeuPheLeuProLysSerValSerAspTyrAspGlyLysLeuSerAsnPheLysThr AlaAlaGluSerPheLysGlyLysIleLeuPheIlePheIleAspSerAspHisThrAsp AsnGlnArgIleLeuGluPhePheGlyLeuLysLysGluGluCysProAlaValArgLeu IleThrLeuGluGluGluMetThrLysTyrLysProGluSerGluGluLeuThrAlaGlu ArgIleThrGluPheCysHisArgPheLeuGluGlyLysIleLysProHisLeuMetSer GlnGluLeuProGluAspTrpAspLysGlnProValLysValLeuValGlyLysAsnPhe GluAspValAlaPheAspGluLysLysAsnValPheValGluPheTyrAlaProTrpCys GlyHisCysLysGlnLeuAlaProIleTrpAspLysLeuGlyGluThrTyrLysAspHis GluAsnIleValIleAlaLysMetAspSerThrAlaAsnGluValGluAlaValLysVal HisSerPheProThrLeuLysPhePheProAlaSerAlaAspArgThrValIleAspTyr AsnGlyGluArgThrLeuAspGlyPheLysLysPheLeuGluSerGlyGlyGlnAspGly AlaGlyAspAspAspAspLeuGluAspLeuGluGluAlaGluGluProAspMetGluGlu AspAspAspGlnLysAlaValLysAspGluLeu. ______________________________________

### Claim 18

A method of producing a transformed microorganism comprising a recombinant vector, wherein said recombinant vector comprises a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of FIG. 3, which nucleotide sequence is operably linked to a promoter sequence, and wherein said recombinant vector replicates and expresses said polypeptide in said transformed microorganism; which method comprises the steps of: (i) transforming a microorganism with said recombinant vector, and (ii) recovering said transformed microorganism.

### Claim 19 (depends on 18)

The method of claim 18, wherein said nucleotide sequence comprises nucleotide 104 to nucleotide 1573 of FIG. 5.

### Claim 20

A method for producing a recombinant vector, wherein said recombinant vector comprises a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of FIG. 3, which nucleotide sequence is operably linked to a promoter sequence, and wherein said recombinant vector replicates and expresses said polypeptide in a microorganism; which method comprises the steps of: (i) inserting an isolated DNA comprising said nucleic acid sequence into a site down stream from a promoter sequence in a vector that replicates in said microorganism so that said nucleic acid sequence is operably linked to said promoter sequence, and (ii) recovering said recombinant vector.

### Claim 21

A method for expressing a biologically active polypeptide, wherein said biologically active polypeptide comprises the amino acid sequence of FIG. 3, which method comprises the step of: cultivating a transformed animal cell or a transformed microorganism belonging to genus Escherichia or genus Saccharomyces, wherein said transformed animal cell or said transformed microorganism is transformed with a recombinant vector, wherein said recombinant vector comprises a nucleotide sequence encoding said biologically active polypeptide, which nucleotide sequence is operably linked to a promoter sequence, and wherein said recombinant vector replicates and expresses said biologically active polypeptide in said animal cell or microorganism.

### Claim 22 (depends on 13)

The method of claim 13, wherein said nucleotide sequence comprises nucleotide 104 to nucleotide 1573 of FIG. 5.

### Claim 23 (depends on 13)

The method of claim 13, wherein said transformed microorganism is Escherichia coli.

### Claim 24 (depends on 13)

The method of claim 13 wherein said transformed microorganism is Escherichia coli MM294/pTB766 (IFO 14611 FERM BP-1842).