TRANSGLUTAMINASE HAVING DISULFIDE BOND INTRODUCED THEREIN

• US 20100143970A1
• Filed: 08/14/2009
• Published: 06/10/2010
• Est. Priority Date: 02/15/2007
• Status: Active Grant

• Current Assignee: Ajinomoto Co., Inc.
• Original Assignee: Ajinomoto Co., Inc.
• Inventors: Suzuki, Eiichiro, Yokoyama, Keiichi, Suzuki, Mototaka, Taguchi, Seiichi, Date, Masayo, Kashiwagi, Tatsuki
• Granted Patent: US 9,062,291 B2
• Time in Patent Office: Days
• US Class Current: 435/69.100
• CPC Class Codes: C12N 9/1044 Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII
• CPC Class Codes: C12Y 203/02013 Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII

Abstract

A transglutaminase protein which is mutated to have improved heat resistance and/or pH stability. A mutation is introduced into WT transglutaminase at a cysteine residue capable of forming a disulfide bond (SS bond).

Claims

1. A protein possessing transglutaminase activity selected from the group consisting of:

(A) a protein comprising the amino acid sequence of SEQ ID NO;

2, but having a mutation selected from the group consisting of;

a) substitution of the amino acids at positions 7 and 58 with cysteine,

b) substitution of the amino acids at positions 46 and 318 with cysteine,

c) substitution of the amino acids at positions 93 and 112 with cysteine,

d) substitution of the amino acids at position 106 and 213 with cysteine,

e) substitution of the amino acids at positions 160 and 228 with cysteine,

f) substitution of the amino acids at positions 2 and 282 with cysteine,

g) substitution of the amino acids at positions 2 and 283 with cysteine,

h) substitution of the amino acids at positions 3 and 283 with cysteine, and

i) substitution of the amino acids at positions 17 and 330 with cysteine;

(B) the protein of (A), but also having one or more substitutions, deletions, additions, and/or insertions of one or more amino acids in said sequence, and wherein said protein has transglutaminase activity;

(C) the protein of (B), which is at least 70% homologous to the amino acid sequence of SEQ ID NO;

2;

(D) a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO;

4, 6, 8, 10, and 12, but wherein said sequence has a mutation selected from the group consisting of;

a) substitution of the amino acids at positions 7 and 58 with cysteine,

b) substitution of the amino acids at positions 46 and 318 with cysteine,

c) substitution of the amino acids at positions 93 and 112 with cysteine,

d) substitution of the amino acids at position 106 and 213 with cysteine,

e) substitution of the amino acids at positions 160 and 228 with cysteine,

f) substitution of the amino acids at positions 2 and 282 with cysteine,

g) substitution of the amino acids at positions 2 and 283 with cysteine,

h) substitution of the amino acids at positions 3 and 283 with cysteine, and

i) substitution of the amino acids at positions 17 and 330 with cysteine;

wherein said positions correspond to those in SEQ ID NO;

2;

(E) the protein of (D), wherein said sequence can also have one or more substitutions deletions, additions, and/or insertions of one or more amino acids in said sequence, and wherein said protein has transglutaminase activity; and

(F) the protein of (E), which is at least 70% homologous to an amino acid sequence selected from the group consisting of SEQ ID NO;

4, 6, 8, 10, and 12.

2. A polynucleotide that encodes the protein of claim 1.

3. A recombinant vector comprising the polynucleotide of claim 2.

4. A host cell transformed with the recombinant vector of claim 3.

5. A method of producing a protein, comprising culturing a host cell which has been transformed with a recombinant vector comprising the polynucleotide of claim 2, and collecting a protein possessing transglutaminase activity.

6. A method of processing a substrate protein, comprising a step selected from the group consisting of

A) allowing the protein of claim 1 to act on the substrate protein,

B) allowing a protein produced by culturing a culturing a host cell which has been transformed with a recombinant vector comprising the polynucleotide which encodes the protein of claim 1 to act on the substrate protein, and

C) allowing a host cell which has been transformed with a recombinant vector comprising the polynucleotide which encodes the protein of claim 1 to act on the substrate protein.

7. The method of claim 6, wherein the processing of the substrate protein is performed at 40°

C. to 100°

C.

8. The method of claim 6, wherein the processing of the substrate protein is performed under conditions involving pH 3 to 4.