Recombinant host for producing L-asparaginase II
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.
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.
13. An isolated DNA molecule encoding a subunit of L-asparaginase II enzyme that comprises SEQ ID NO: 2.
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.