Plasmid Vector

• US 20110021751A1
• Filed: 03/24/2009
• Published: 01/27/2011
• Est. Priority Date: 03/25/2008
• Status: Active Grant

• Current Assignee: KAO Corporation
• Original Assignee: KAO Corporation
• Inventors: Takimura, Yasushi, Sumitomo, Nobuyuki, Izawa, Yoshifumi
• Granted Patent: US 8,389,685 B2
• Time in Patent Office: Days
• US Class Current: 530/350
• CPC Class Codes: C07K 14/32 from Bacillus (G)
• CPC Class Codes: C12N 15/67 General methods for enhancing the expression

Abstract

To provide a plasmid vector having high productivity which can be used for large scale production of an industrially valuable and useful protein, and a transformant using the plasmid vector. A plasmid vector having DNA which encodes a plasmid replication protein in which one or more amino acid residues that are selected from (a) position 48, (b) position 262, (c) position 149 and (d) position 198 in the amino acid sequence represented by SEQ ID NO: 2 are substituted with (a) Ala, Gly, Thr, Arg, Glu, Asn or Gln, (b) Gly, Ser, Thr, Cys or Val, (c) Asn, and (d) Glu, respectively.

Claims

1. A plasmid vector comprising DNA which encodes a plasmid replication protein, wherein the replication protein comprises substitution of one or more amino acid residues that are selected from (a) position 48, (b) position 262, (c) position 149 and (d) position 198 in the amino acid sequence represented by SEQ ID NO:

2 with the following amino acid residue;

(a) position;

Ala, Gly, Thr, Arg, Glu, Asn or Gln

(b) position;

Gly, Ser, Thr, Cys or Val

(c) position;

Asn

(d) position;

Glu.

3. The plasmid vector according to claim 1 or 2, further comprising a nucleotide sequence of a promoter region and a secretory signal region of a gene which encodes alkaline cellulase derived from a microorganism belonging to Bacillus spp.

4. The plasmid vector according to claim 3, wherein the nucleotide sequence of a promoter region and a secretory signal region of a gene which encodes alkaline cellulase derived from a microorganism belonging to Bacillus spp. is a nucleotide sequence including base numbers 1 to 659 of cellulase gene which consists of a nucleotide sequence represented by SEQ ID NO:

13, a nucleotide sequence including base numbers 1 to 696 of cellulase gene which consists of a nucleotide sequence represented by SEQ ID NO;

14, a nucleotide sequence having at least 70% identity to the any one of the nucleotide sequences, or a nucleotide sequence in which part of the nucleotide sequence is deleted.

5. A transformant comprising the plasmid vector according to claim 3.

6. The transformant according to claim 5, wherein a host of the plasmid vector is a microorganism.

12. A transformant comprising the plasmid vector according to claim 4.

13. The transformant according to claim 12, wherein a host of the plasmid vector is a microorganism.

2. A plasmid vector comprising DNA which encodes a plasmid replication protein, wherein the replication protein comprises substitution of one or more amino acid residues that are selected from the amino acid residues present at positions corresponding to each of (a) position 48, (b) position 262, (c) position 149 and (d) position 198 of SEQ ID NO:

2 in an amino acid sequence having at least 80% identity to the amino acid sequence represented by SEQ ID NO;

2 with the following amino acid residue;

(a) position;

Ala, Gly, Thr, Arg, Glu, Asn or Gln

(b) position;

Gly, Ser, Thr, Cys or Val

(c) position;

Asn

(d) position;

Glu.

7. A method for producing a polypeptide comprising the steps of:

constructing a plasmid vector comprising DNA which encodes a target polypeptide and DNA which encodes a plasmid replication protein, wherein the replication protein comprises substitution of one or more amino acid residues that are selected from (a) position 48, (b) position 262, (c) position 149 and (d) position 198 in the amino acid sequence represented by SEQ ID NO;

2 with the following amino acid residue,

(a) position;

Ala, Gly, Thr, Arg, Glu, Asn or Gln

(b) position;

Gly, Ser, Thr, Cys or Val

(c) position;

Asn

(d) position;

Glu;

transforming a host microorganism with the plasmid vector; and

culturing the host microorganism and collecting the target polypeptide produced therefrom.

9. The method according to claim 7 or 8, wherein the plasmid vector further comprises a nucleotide sequence of a promoter region and a secretory signal region of a gene which encodes alkaline cellulase derived from a microorganism belonging to Bacillus spp.

10. The method according to claim 9, wherein the nucleotide sequences of a promoter region and a secretory signal region of a gene which encodes alkaline cellulase derived from a microorganism belonging to Bacillus spp. is a nucleotide sequence including base numbers 1 to 659 of cellulase gene which consists of a nucleotide sequence represented by SEQ ID NO:

13, a nucleotide sequence including base numbers 1 to 696 of cellulase gene which consists of a nucleotide sequence represented by SEQ ID NO;

14, a nucleotide sequence having at least 70% identity to any one of the nucleotide sequences, or a nucleotide sequence in which part of the nucleotide sequence is deleted.

11. A polypeptide which is produced according to the method of claim 7 or 8.

8. A method for producing a polypeptide comprising the steps of:

constructing a plasmid vector comprising DNA which encodes a target polypeptide and DNA which encodes a plasmid replication protein comprising substitution of one or more amino acid residues that are selected from the amino acid residues present at positions corresponding to each of (a) position 48, (b) position 262, (c) position 149 and (d) position 198 of SEQ ID NO;

2 in an amino acid sequence having at least 80% identity to the amino acid sequence represented by SEQ ID NO;

2 with the following amino acid residue,

(a) position;

Ala, Gly, Thr, Arg, Glu, Asn or Gln

(b) position;

Gly, Ser, Thr, Cys or Val

(c) position;

Asn

(d) position;

Glu;

transforming a host microorganism with the plasmid vector; and

culturing the host microorganism and collecting the target polypeptide produced therefrom.