RECOMBINANT HOST FOR PRODUCING L-ASPARAGINASE II

• US 20100273236A1
• Filed: 05/04/2010
• Published: 10/28/2010
• Est. Priority Date: 06/30/2006
• Status: Abandoned Application

• Current Assignee: Servier IP UK Ltd. (Baxter International Inc.)
• Original Assignee: Defiante Farmaceutica S.A. (Alfasigma S.p.A.)
• Inventors: WANG, Maoliang, FILPULA, David Ray
• Application Number:
  US12/773,320
• Publication Number:
  US 20100273236A1
• Time in Patent Office: Days
• 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 L-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.

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 recombinant extrachromosomal vector comprises a DNA molecule encoding the L-asparaginase protein, that 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. 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 1, and isolating the produced L-asparaginase II enzyme.