Fusion protein-bound magnetic particles for recombinant production and magnetic separation of polypeptides of interest

• US 5,861,285 A
• Filed: 09/18/1995
• Issued: 01/19/1999
• Est. Priority Date: 09/16/1994
• Status: Expired due to Term

• Current Assignee: National University Corporation Tokyo University of Agriculture and Technology
• Original Assignee: TDK Corporation
• Inventors: Matsunaga, Tadashi
• Primary Examiner(s): Fitzgerald, David L.
• Application Number:
  US08/529,600
• Time in Patent Office: 1,219 Days
• Field of Search: 435/69.7, 435/252.3, 435/320.1, 536/23.4, 536/23.7, 530/427, 530/350
• US Class Current: 435/69.7
• CPC Class Codes: C07K 14/195 from bacteria
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 11/00 Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins
• CPC Class Codes: G01N 2333/195 from bacteria
• CPC Class Codes: G01N 33/54326 Magnetic particles
• CPC Class Codes: G01N 33/554 the carrier being a biological cell or cell fragment, e.g. bacteria, yeast cells

Abstract

A fusion DNA sequence, which is obtained by fusing a gene coding for another useful protein to a fragment of a magA gene coding for a protein bound to an organic membrane for covering magnetic particles produced in cells of a magnetic bacterium AMB-1, is expressed in the magnetic bacterium to obtain the protein in a state of being bound to the magnetic particles. According to the present invention, useful proteins such as enzymes and antibodies can be stably obtained in a state of being bound to the organic membrane of the magnetic particles only by cultivating a transformed magnetic bacterium, and separating the magnetic particles produced in cells, without any necessity to perform a treatment such as immobilization. The functional protein immobilized on the magnetic particles can be magnetically controlled. Thus the function can be efficiently exhibited at a desired topical position. Magnetic particles to which a desired protein is bound can be semipermanently produced only by maintaining and cultivating an identical bacterial strain. Since the protein is produced on the magnetic particles, an objective protein can be magnetically separated and recovered in a short period of time. Thus it is possible to perform efficient separation and purification.

Claims

1. A fusion protein comprising:

(a) a fragment of the magA gene product, the fragment comprising the amino acid sequence shown as residues 7-380 of SEQ ID NO;

2,

(b) the amino acid sequence of a first protein of interest, and

(c) optionally, the amino acid sequence of a second protein of interest;

wherein the first and optional second proteins of interest are the same or different, and wherein the first and optional second proteins of interest are joined to the N- or C-terminal of the fragment of the magA gene product.

2. A protein-bound magnetic particle comprising a magnetic particle, an organic membrane covering the surfgace of the magnetic particle, and a fusion protein according to claim 1 bound to the organic membrane.

3. A protein-bound magnetic particle according to claim 2, wherein either or both of the first and second proteins of interest is independently selected from the group consisting of an immunoglobulin, a binding protein, and an enzyme.

4. A DNA molecule encoding a fusion protein according to claim 1.

5. A plasmid comprising the nucleotide sequence of a DNA molecule according to claim 4.

6. A transformed host magnetic bacterium comprising a plasmid according to claim 5.

7. A transformed host magnetic bacterium according to claim 6, wherein the bacterium belongs to the species Magnetospirillum or Desulfovibrio.

8. A process for producing a protein-bound magnetic particle comprising a magnetic particle, an organic membrane covering the surface of the magnetic particle, and a fusion protein,

the process comprising cultivating a transformed host magnetic bacterium according to claim 6 under conditions suitable to effect expression of the DNA molecule contained in the plasmid and to effect synthesis of the protein-bound magnetic particle by the transformed host magnetic bacterium.

9. A process according to claim 8, further comprising recovering the protein-bound magnetic particle by magnetic separation.

10. A fusion protein comprising:

(a) a fragment of the magA gene product, the fragment comprising the amino acid sequence shown as residues 7-380 of SEQ ID NO;

2, and

(b) the amino acid sequence of a protein of interest selected from the group consisting of an immunoglobulin, a binding protein, and an enzyme;

wherein the protein of interest is joined to the N- or C-terminal of the fragment of the magA gene product.

11. A protein-bound magnetic particle comprising a magnetic particle, an organic membrane covering the surface of the magnetic particle, and a fusion protein according to claim 10 bound to the organic membrane.

12. A DNA molecule encoding a fusion protein according to claim 10.

13. A plasmid comprising the nucleotide sequence of a DNA molecule according to claim 12.

14. A transformed host magnetic bacterium comprising a plasmid according to claim 13.

15. A transformed host magnetic bacterium according to claim 14, wherein the bacterium belongs to the species Magnetospirillum or Desulfovibrio.

16. A process for producing a protein-bound magnetic particle comprising a magnetic particle, an organic membrane covering the surface of the magnetic particle, and a fusion protein,

the process comprising cultivating a transformed host magnetic bacterium according to claim 14 under conditions suitable to effect expression of the DNA molecule contained in the plasmid and to effect synthesis of the protein-bound magnetic particle by the transformed host magnetic bacterium.

17. A process according to claim 16, further comprising recovering the protein-bound magnetic particle by magnetic separation.