Process for the production and purification of proteins and the production of arrays of proteins

• US 20040023329A1
• Filed: 07/25/2003
• Published: 02/05/2004
• Est. Priority Date: 12/20/2000
• Status: Active Grant

• Current Assignee: Altana Pharma AG (Takeda Pharmaceutical Company Limited)
• Original Assignee: Altana Pharma AG (Takeda Pharmaceutical Company Limited)
• Inventors: Schafer, Klaus Peter, Muhldorfer, Ingeborg
• Granted Patent: US 7,202,055 B2
• Time in Patent Office: Days
• US Class Current: 435/69.1
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli

Abstract

Described is a novel process for the production of at least one protein of interest by secretion of the protein of interest from a pro- or eukaryotic host cell in a compartment system, which host cell is stably expressing a secretion system and capable of heterologous secretion of the protein of interest and which compartment system has at least a first and a second compartment and wherein the host cell is located in the first compartment and wherein the first and second compartment are separated from each other by a barrier, wherein the barrier is permeable for the secreted protein of interest, but not permeable for the host cell.

Claims

1. Process for the production of at least one protein of interest by secretion of the protein of interest from a host cell in a compartment system, which host cell is stably expressing a secretion system and capable of heterologous secretion of the protein of interest and which compartment system has at least a first and a second compartment and wherein the host cell is located in the first compartment and wherein the first and second compartment are separated from each other by a barrier, wherein the barrier is permeable for the secreted protein of interest, but not permeable for the host cell.

2. Process according to claim 1 wherein the host cell is expressing a secretion system encoded on a mobile vector.

3. Process according to claim 2, wherein the mobile vector is either a plasmid or a cosmid.

4. Process according to claims 1 and 2, wherein the barrier is part of the first compartment of the compartment system.

5. Process according to claims 1 and 2, wherein the barrier is a membrane, which membrane filter has a pore diameter that prevents host cell migration and allows the diffusion of the secreted protein of interest through the membrane.

6. Process according to claims 1 and 2, wherein the first compartment is located horizontally above the second compartment and the secreted protein of interest will diffuse by means of gravity through the barrier from the first compartment to the second compartment.

7. Process according to claim 5, wherein the host cell is located on the surface of the filter and the second compartment is a solid phase attached to the opposite surface of the filter and which solid phase is capable of retaining the secreted protein after diffusion through the filter.

8. Process according to claims 1 and 2, wherein the host cell is either E. coli or Pichia patoris.

9. Process according to claims 1 and 2, wherein the secretion of the protein of interest is effected by growth of the host cell.

10. Process according to claim 5, wherein the first and second compartment are stacked microwells which are separated by said filter.

11. Process according to claims 1 and 2, wherein the protein of interest is obtained as a spot in the second compartment or wherein the protein of interest is obtained in solution in the second compartment.

12. Process according to claims 1 and 2, wherein the secreted protein of interest carries an affinity tag, an epitope tag and/or carries a protease cleavage site suitable for removal of secretion signal and/or tag sequences.

13. Process according to claims 1 and 2 for the production of several different proteins of interest, wherein each host cell secreting a respective protein of interest is located in a defined area of the first compartment and each of the secreted proteins of interest will migrate to defined area of the second compartment or wherein each host cell secreting a respective protein of interest is located in different first compartments and each of the secreted proteins of interest will migrate to different second compartments.

14. Process for the production of an array of several different proteins of interest, by secretion of the proteins of interest from respective host cells in a compartment system, which host cells are stably expressing a secretion system, which enables the host cells to secrete heterologous the respective protein of interest and which compartment system has at least a first and a second compartment and wherein the different host cells are arranged in the form of an array in said first compartment and wherein the first and second compartment are separated from each other by a barrier, wherein the barrier is permeable for the secreted proteins of interest, but not permeable for the host cell and wherein the secreted proteins of interest are received in the second compartment in the form of an array.

15. Process according to claim 14 wherein the barrier is part of the first compartment of the compartment system.

16. Process according to claim 14 wherein the array of proteins corresponds to the proteom of an organism of interest.

17. E.coli strain harbouring the hemolysin secretion system genes hlyB and hlyD and upstream sequences within the chromosomal lacZ gene.