US 12,509,665 B2Granted

Thioredoxin mutant, preparation method thereof, and application thereof in production of recombinant fusion protein

AssigneeChengdu Enzpro Biotechnology Co., Ltd

InventorYi Liu

Life of this patent

Estimating the term…

Abstract

Disclosed are a thioredoxin mutant, preparation method thereof, and application thereof in production of recombinant fusion protein. The thioredoxin mutant is prepared by modifying at least one amino acid in the amino acid sequence of SEQ ID NO: 1.

Claims1

Dependent claims

A thioredoxin mutant having the amino acid sequence of SEQ ID NO:

1 that consists of a plurality of point mutations, wherein the plurality of point mutations is selected from the group consisting of T15I+K51R;
D23T+K51R;
D85E+K93P;
T15I+K93P;
D23T+K93P;
K51R+K102H;
T15I+K102H;
T15I+D23T;
D23T+D85E+K93P;
T15I+D85E+K93P;
K51R+D85E+K93P;
R44Q+D85E+K93P;
D85E+K93P+K102H;
D23T+R440+K51R;
R44Q+K51R+D85E;
D23T+K51R+D85E+K93P;
T15I+K51R+D85E+K93P;
T15I+D23T+D85E+K93P;
D23T+R44Q+K51R+D85E;
R44Q+K51R+D85E+K93P;
D23T+R44Q+D85E+K93P;
T15I+D23T+K51R+D85E+K93P;
D23T+K51R+D85E+K93P+K102H;
D23T+R44Q+K51R+D85E+K93P;
T15I+R44Q+K51R+D85E+K93P;
A12I+N56K+S71M+K93P+K102H;
A12I+T15I+N56K+S71M+K102H;
T15I+D23T+C29S+R44Q+K51R+D85E;
T15I+D23T+R44Q+K51R+D85E+K93P;
A12I+D23T+N56K+S71M+K93P+K102H;
A12I+N56K+S71M+D85E+K93P+K102H;
T15I+D23T+R44Q+K51R+D85E+K93P;
D23T+R44Q+K51R+D85E+K93P+K102H;
T15I+D23T+R44Q+K51R+D85E+K93P+K102H;
T15I+D23T+R44Q+K51R+N56K+D85E+K93P;
T15I+D23T+R44Q+K51R+S71M+D85E+K93P;
A12I+T15I+D23T+R44Q+K51R+D85E+K93P;
A12I+T15I+D23T+R44Q+K51R+D85E+K93P+K102H;
T15I+D23T+R44Q+K51R+N56K+D85E+K93P+K102H;
T15I+D23T+R44Q+K51R+S71M+D85E+K93P+K102H;
T15I+D23T+C29S+R44Q+K51R+D85E+K93P+K102H;
A12I+T15I+D23T+R44Q+K51R+N56K+D85E+K93P;
T15I+D23T+C29S+R44Q+K51R+N56K+S71M+D85E;
A12I+T15I+D23T+R44Q+K51R+N56K+S71M+D85E+K93P;
T15I+D23T+D43E+R44Q+K51R+T74I+D85E+Q91K+K93P;
T15I+D23T+C29S+R44Q+K51R+S71M+D85E+K93P+K102H;
T15I+D23T+R44Q+K51R+N56K+S71M+D85E+K93P+K102H;
A12I+T15I+D23T+R44Q+K51R+S71M+D85E+K93P+K102H;
A12I+T15I+D23T+C29S+R44Q+K51R+N56K+S71M+D85E+K93P;
T15I+D23T+D43E+R44Q+K51R+S71M+T74I+D85E+Q91K+K93P;
T15I+D23T+D43E+R44Q+K51R+T74I+D85E+Q91K+K93P+K102H;
A12I+T15I+D23T+R44Q+K51R+N56K+S71M+D85E+K93P+K102H;
A12I+T15I+D23T+R44Q+K51R+N56K+S71M+D85E+Q91K+K93P+K102H;
A12I+T15I+D23T+C29S+R44Q+K51R+N56K+S71M+D85E+K93P+K102H;
A12I+T15I+D23T+R44Q+K51R+N56K+S71M+T74I+D85E+K93P+K102H;
A12I+T15I+D23T+D43E+R44Q+K51R+N56K+S71M+D85E+K93P+K102H;
A12I+T15I+D23T+C29S+R44Q+K51R+N56K+S71M+D85E+Q91K+K93P+K102H;
A12I+T15I+D23T+R44Q+K51R+N56K+S71M+T74I+D85E+Q91K+K93P+K102H;
A12I+T15I+D23T+D43E+R44Q+K51R+N56K+S71M+D85E+Q91K+K93P+K102H;
A12I+T15I+D23T+D43E+R44Q+K51R+N56K+S71M+T74I+D85E+K93P+K102H;
A12I+T15I+D23T+C29S+R44Q+K51R+N56K+S71M+T74I+D85E+Q91K+K93P+K102H;
A12I+T15I+D23T+D43E+R44Q+K51R+N56K+S71M+T74I+D85E+Q91K+K93P+K102H;
A12I+T15I+D23T+R44Q+K51R+N56K+S71M+T74I+Q82K+D85E+Q91K+K93P+K102H;
A12I+T15I+D23T+R44Q+K51R+N56K+D58E+S71M+T74I+D85E+Q91K+K93P+K102H;
A12I+T15I+D23T+D43E+R44Q+K51R+N56K+S71M+T74I+Q82K+D85E+Q91K+K93P+K102H;
A12I+T15I+D23T+C29S+D43E+R44Q+K51R+N56K+S71M+T74I+D85E+Q91K+K93P+K102H;
A12I+A13I+T15I+D23T+D43E+R44Q+K51R+N56K+S71M+T74I+D85E+Q91K+K93P+K102H;
A12I+T15I+D23T+R33K+D43E+R44Q+K51R+N56K+S71M+T74I+D85E+Q91K+K93P+K102H;
A12I+T15I+D23T+D43E+R44Q+K51R+N56K+D58E+S71M+T74I+D85E+Q91K+K93P+K102H;
A12I+A13I+T15I+D23T+C29S+D43E+R44Q+K51R+N56K+S71M+T74I+D85E+Q91K+K93P+K102H;
A12I+A13I+T15I+D23T+R33K+D43E+R44Q+K51R+N56K+S71M+T74I+D85E+Q91K+K93P+K102H;
A12I+T15I+D23T+D43E+R44Q+K51R+N56K+D58E+S71M+T74I+Q82K+D85E+Q91K+K93P+K102H;
A12I+A13I+T15I+D23T+C29S+D43E+R44Q+K51R+N56K+D58E+S71M+T74I+D85E+Q91K+K93P+K102H;
A12I+A13I+T15I+D23T+R33K+D43E+R44Q+K51R+N56K+D58E+S71M+T74I+D85E+Q91K+K93P+K102H;
A12I+T15I+D23T+R33K+D43E+R44Q+K51R+N56K+D58E+S71M+T74I+Q82K+D85E+Q91K+K93P+K102H;
A12I+A13I+T15I+D23T+R33K+D43E+R44Q+K51R+N56K+D58E+S71M+T74I+Q82K+D85E+Q91K+K93P+K102H;
A12I+A13I+T15I+D23T+C29S+R33K+D43E+R44Q+K51R+N56K+D58E+S71M+T74I+D85E+Q91K+K93P+K102H;
A12I+A13I+T15I+D23T+C29S+D43E+R44Q+K51R+N56K+D58E+S71M+T74I+Q82K+D85E+Q91K+K93P+K102H;
and A12I+A13I+T15I+D23T+C29S+R33K+D43E+R44Q+K51R+N56K+D58E+S71M+T74I+Q82K+D85E+Q91K+K93P+K102H.

76 more elements

Dependencies are inferred from the claim text.

Facts

Application: 17/597,140Filed 8 Jul 2019
Priority: CN 201910557170.225 Jun 2019
Published: US 2022/0411764 A129 Dec 2022
Granted: 30 Dec 2025
Term ends: Not estimatedThe source records don’t support an estimate.

Classification7 codes

C12N 9/0051
Oxidoreductases: acting on a sulfur group of donors
C07K 14/605
Hormones: glucagons
C12Y 108/01008
With NAD+ or NADP+ as acceptor: protein-disulfide reductase , i.e. thioredoxin
C07K 2319/35
Fusion polypeptide: containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin

Family

US 2022/0411764 A1Pre-grant publication · published 29 Dec 2022
US 12,509,665 B2This patent

From the US filing history. Foreign family members aren’t listed.

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Abstract

A thioredoxin mutant having the amino acid sequence of SEQ ID NO:

1 that consists of a plurality of point mutations, wherein the plurality of point mutations is selected from the group consisting of T15I+K51R;
D23T+K51R;
D85E+K93P;
T15I+K93P;
D23T+K93P;
K51R+K102H;
T15I+K102H;
T15I+D23T;
D23T+D85E+K93P;
T15I+D85E+K93P;
K51R+D85E+K93P;
R44Q+D85E+K93P;
D85E+K93P+K102H;
D23T+R440+K51R;
R44Q+K51R+D85E;
D23T+K51R+D85E+K93P;
T15I+K51R+D85E+K93P;
T15I+D23T+D85E+K93P;
D23T+R44Q+K51R+D85E;
R44Q+K51R+D85E+K93P;
D23T+R44Q+D85E+K93P;
T15I+D23T+K51R+D85E+K93P;
D23T+K51R+D85E+K93P+K102H;
D23T+R44Q+K51R+D85E+K93P;
T15I+R44Q+K51R+D85E+K93P;
A12I+N56K+S71M+K93P+K102H;
A12I+T15I+N56K+S71M+K102H;
T15I+D23T+C29S+R44Q+K51R+D85E;
T15I+D23T+R44Q+K51R+D85E+K93P;
A12I+D23T+N56K+S71M+K93P+K102H;
A12I+N56K+S71M+D85E+K93P+K102H;
T15I+D23T+R44Q+K51R+D85E+K93P;
D23T+R44Q+K51R+D85E+K93P+K102H;
T15I+D23T+R44Q+K51R+D85E+K93P+K102H;
T15I+D23T+R44Q+K51R+N56K+D85E+K93P;
T15I+D23T+R44Q+K51R+S71M+D85E+K93P;
A12I+T15I+D23T+R44Q+K51R+D85E+K93P;
A12I+T15I+D23T+R44Q+K51R+D85E+K93P+K102H;
T15I+D23T+R44Q+K51R+N56K+D85E+K93P+K102H;
T15I+D23T+R44Q+K51R+S71M+D85E+K93P+K102H;
T15I+D23T+C29S+R44Q+K51R+D85E+K93P+K102H;
A12I+T15I+D23T+R44Q+K51R+N56K+D85E+K93P;
T15I+D23T+C29S+R44Q+K51R+N56K+S71M+D85E;
A12I+T15I+D23T+R44Q+K51R+N56K+S71M+D85E+K93P;
T15I+D23T+D43E+R44Q+K51R+T74I+D85E+Q91K+K93P;
T15I+D23T+C29S+R44Q+K51R+S71M+D85E+K93P+K102H;
T15I+D23T+R44Q+K51R+N56K+S71M+D85E+K93P+K102H;
A12I+T15I+D23T+R44Q+K51R+S71M+D85E+K93P+K102H;
A12I+T15I+D23T+C29S+R44Q+K51R+N56K+S71M+D85E+K93P;
T15I+D23T+D43E+R44Q+K51R+S71M+T74I+D85E+Q91K+K93P;
T15I+D23T+D43E+R44Q+K51R+T74I+D85E+Q91K+K93P+K102H;
A12I+T15I+D23T+R44Q+K51R+N56K+S71M+D85E+K93P+K102H;
A12I+T15I+D23T+R44Q+K51R+N56K+S71M+D85E+Q91K+K93P+K102H;
A12I+T15I+D23T+C29S+R44Q+K51R+N56K+S71M+D85E+K93P+K102H;
A12I+T15I+D23T+R44Q+K51R+N56K+S71M+T74I+D85E+K93P+K102H;
A12I+T15I+D23T+D43E+R44Q+K51R+N56K+S71M+D85E+K93P+K102H;
A12I+T15I+D23T+C29S+R44Q+K51R+N56K+S71M+D85E+Q91K+K93P+K102H;
A12I+T15I+D23T+R44Q+K51R+N56K+S71M+T74I+D85E+Q91K+K93P+K102H;
A12I+T15I+D23T+D43E+R44Q+K51R+N56K+S71M+D85E+Q91K+K93P+K102H;
A12I+T15I+D23T+D43E+R44Q+K51R+N56K+S71M+T74I+D85E+K93P+K102H;
A12I+T15I+D23T+C29S+R44Q+K51R+N56K+S71M+T74I+D85E+Q91K+K93P+K102H;
A12I+T15I+D23T+D43E+R44Q+K51R+N56K+S71M+T74I+D85E+Q91K+K93P+K102H;
A12I+T15I+D23T+R44Q+K51R+N56K+S71M+T74I+Q82K+D85E+Q91K+K93P+K102H;
A12I+T15I+D23T+R44Q+K51R+N56K+D58E+S71M+T74I+D85E+Q91K+K93P+K102H;
A12I+T15I+D23T+D43E+R44Q+K51R+N56K+S71M+T74I+Q82K+D85E+Q91K+K93P+K102H;
A12I+T15I+D23T+C29S+D43E+R44Q+K51R+N56K+S71M+T74I+D85E+Q91K+K93P+K102H;
A12I+A13I+T15I+D23T+D43E+R44Q+K51R+N56K+S71M+T74I+D85E+Q91K+K93P+K102H;
A12I+T15I+D23T+R33K+D43E+R44Q+K51R+N56K+S71M+T74I+D85E+Q91K+K93P+K102H;
A12I+T15I+D23T+D43E+R44Q+K51R+N56K+D58E+S71M+T74I+D85E+Q91K+K93P+K102H;
A12I+A13I+T15I+D23T+C29S+D43E+R44Q+K51R+N56K+S71M+T74I+D85E+Q91K+K93P+K102H;
A12I+A13I+T15I+D23T+R33K+D43E+R44Q+K51R+N56K+S71M+T74I+D85E+Q91K+K93P+K102H;
A12I+T15I+D23T+D43E+R44Q+K51R+N56K+D58E+S71M+T74I+Q82K+D85E+Q91K+K93P+K102H;
A12I+A13I+T15I+D23T+C29S+D43E+R44Q+K51R+N56K+D58E+S71M+T74I+D85E+Q91K+K93P+K102H;
A12I+A13I+T15I+D23T+R33K+D43E+R44Q+K51R+N56K+D58E+S71M+T74I+D85E+Q91K+K93P+K102H;
A12I+T15I+D23T+R33K+D43E+R44Q+K51R+N56K+D58E+S71M+T74I+Q82K+D85E+Q91K+K93P+K102H;
A12I+A13I+T15I+D23T+R33K+D43E+R44Q+K51R+N56K+D58E+S71M+T74I+Q82K+D85E+Q91K+K93P+K102H;
A12I+A13I+T15I+D23T+C29S+R33K+D43E+R44Q+K51R+N56K+D58E+S71M+T74I+D85E+Q91K+K93P+K102H;
A12I+A13I+T15I+D23T+C29S+D43E+R44Q+K51R+N56K+D58E+S71M+T74I+Q82K+D85E+Q91K+K93P+K102H;
and A12I+A13I+T15I+D23T+C29S+R33K+D43E+R44Q+K51R+N56K+D58E+S71M+T74I+Q82K+D85E+Q91K+K93P+K102H.

76 more elements

Facts

Application

17/597,140Filed 8 Jul 2019

Priority

CN 201910557170.225 Jun 2019

Published

US 2022/0411764 A129 Dec 2022

Granted

30 Dec 2025

Term ends

Not estimatedThe source records don’t support an estimate.

Classification7 codes

C12N 9/0051
Oxidoreductases: acting on a sulfur group of donors
C07K 14/605
Hormones: glucagons
C12Y 108/01008
With NAD+ or NADP+ as acceptor: protein-disulfide reductase , i.e. thioredoxin
C07K 2319/35
Fusion polypeptide: containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin