Recombinant host for producing L-asparaginase II

• US 7,807,436 B2
• Filed: 06/08/2007
• Issued: 10/05/2010
• Est. Priority Date: 06/30/2006
• Status: Active Grant

• Current Assignee: Servier IP UK Ltd. (Baxter International Inc.)
• Original Assignee: Defiante Farmaceutica S.A.
• Inventors: Wang, Maoliang, Filpula, David Ray
• Primary Examiner(s): Ramirez; Delia M
• Application Number:
  US11/759,988
• Publication Number:
  US 20080063632A1
• Time in Patent Office: 1,215 Days
• Field of Search: None
• US Class Current: 435/229
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C12N 9/82 Asparaginase (3.5.1.1)
• CPC Class Codes: C12N 9/96 Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention provides a recombinant Escherichia coli host cell for producing an Escherichia coli-asparaginase II enzyme. The host cell includes an Escherichia coli chromosome and at least one copy of a recombinant extrachromosomal vector, wherein the recombinant extrachromosomal vector encodes the L-asparaginase II enzyme, wherein the host cell chromosome also encodes the same L-asparaginase II enzyme, and wherein the host chromosome does not encode any other isoform of L-asparaginase II.

Claims

1. A recombinant Escherichia coli host cell for producing an Escherichia coli L-asparaginase II enzyme, comprising an Escherichia coli host cell chromosome and at least one copy of a recombinant extrachromosomal vector, wherein the recombinant extrachromosomal vector encodes a subunit of the L-asparaginase II enzyme, wherein the host cell chromosome also encodes the same subunit of the L-asparaginase II enzyme, and wherein the host cell chromosome does not encode any other isoform of L-asparaginase II, wherein the encoded. L-asparaginase II subunit comprises SEQ ID NO:

1; and

the recombinant extrachromosomal vector comprises a DNA molecule comprising SEQ ID NO;

2.

2. The recombinant Escherichia coli host cell of claim 1, wherein the extrachromosomal vector is a plasmid.

3. The recombinant Escherichia coli host cell of claim 1 wherein the DNA molecule is operatively connected to a suitable promoter.

4. The recombinant Escherichia coli host cell of claim 3 wherein the promoter is selected from the group consisting of T7, araB, P_R/P_L, phoA, trc, and trp promoters.

5. The recombinant Escherichia coli host cell of claim 3 wherein the recombinant extrachromosomal vector further comprises an operator, ribosome binding site, signal sequence, transcriptional terminator, antibiotic selection marker, origin of replication, and a regulated copy of the repressor.

6. The recombinant Escherichia coli host cell of claim 3 wherein the host cell chromosome comprises a DNA molecule according to SEQ ID NO:

3.

7. An isolated nucleic acid molecule encoding an L-asparaginase II enzyme subunit of SEQ ID NO:

1, wherein the isolated nucleic acid molecule comprises a nucleic acid selected from the group consisting of SEQ ID NO;

2 and SEQ ID NO;

3.

8. A recombinant extrachromosomal vector, wherein the recombinant extrachromosomal vector comprises SEQ ID NO:

2, and encodes a subunit of the L-asparaginase II enzyme.

9. The recombinant extrachromosomal vector of claim 8 wherein the vector is a plasmid.

10. The recombinant extrachromosomal vector of claim 8 wherein the vector is plasmid pEN537.

11. An Escherichia coli host cell comprising the plasmid of claim 10, that is designated as EN538 and deposited as ATCC Number PTA 7490.

12. A method of producing a recombinant L-asparaginase II enzyme substantially free of other L-asparaginase II isomers, comprising culturing the host cell of claim 11, and isolating the produced L-asparaginase II enzyme.

14. The recombinant extrachromosomal vector of claim 8 wherein SEQ ID NO:

2 is operatively connected to a suitable promoter.

15. The recombinant extrachromosomal vector of claim 14 wherein the promoter is selected from the group consisting of T7, araB, P_R/P_L, phoA, trc, and trp promoters.

13. An isolated DNA molecule encoding a subunit of L-asparaginase II enzyme that comprises SEQ ID NO:

2.