# US 5,700,659 A · Polypeptide possessing protein disulfide isomerase activity gene encoding the same and process for producing the same

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- Number: US 5,700,659 A
- Document ID: US5700659A
- Status: Granted
- Kind: A
- Application number: 08/464,365
- Filed: 1995-06-05
- Granted: 1997-12-23
- Earliest claimed: 1992-05-27
- Assignees: Kabushiki Kaisha Toyota Chuo Kenkusho
- Inventors: Yukio Yamada; Osamu Asami; Hidehiko Sugiyama; Chie Idekoba; Fumihiko Hoshino; Masana Hirai; Tsutomu Kajino; Takao Imaeda; Kiyoko Sarai
- Web page: https://patentgrep.com/patent/US5700659A

## CPC

- C12N 9/90: Isomerases (5.)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

A highly thermostable polypeptide possessing protein disulfide isomerase (PDI) activity, a gene coding for the polypeptide and a process for producing the polyeptide are provided. The polypeptide possessing PDI activity is characterized by A) having a capability of catalyzing a disulfide exchange in proteins, B) recognizing mainly ribonuclease A as a substrate, C) having a suitable active temperature of 20.degree. to 70.degree. C., D) being stable at a pH value of 6 to 9, and E) having a molecular weight of about 60,000 to 62,000. Since it has a higher thermostability and exhibits a stable activity in a wider dithiothreitol concentration range as compared with the conventional PDI, it is possible to provide a novel enzyme active protein which can be advantageously used for a refolding reaction of certain proteins. Further, a process which enables the polylpeptide possessing PDI activity to be efficiently produced using Humicola insolens or a transformant transformed with an expression vector containing the above-described gene is also provided.

## Claims

### Claim 1

An isolated gene coding for the amino acid sequence of SEQ ID NO: 1.

### Claim 2 (depends on 1)

A transformant which has been transformed with a vector containing said gene according to claim 1.

### Claim 3 (depends on 2)

A process for producing a highly thermostable polypeptide possessing protein disulfide activity, which comprises the steps of:
(A) culturing the transformant of claim 2 in a nutrient media, and
(B) recovering said polypeptide possessing protein disulfide isomerase from the media.

### Claim 4

A process for producing a highly thermostable polypeptide possessing protein disulfide isomerase activity, which comprises culturing Humicola insolens KASI in a nutrient medium to produce said polypeptide and recovering said polypeptide from said medium,
wherein said polypeptide has the following properties:
A) it catalyzes a disulfide exchange in proteins;
B) ribonuclease A is a substrate;
C) it has a suitable active temperature of 20.degree.to 70.degree. C.;
D) it is stable at a pH value of 6 to 9; and
E) it has a molecular weight of about 60,000 to 62,000 as measured by SDS-polyacrylamide gel electrophoresis.