Production and use of multimeric hemoglobins

• US 6,184,356 B1
• Filed: 04/13/1998
• Issued: 02/06/2001
• Est. Priority Date: 05/10/1989
• Status: Expired due to Fees

• Current Assignee: Baxter Biotech Technology SARL (Baxter International Inc.)
• Original Assignee: Baxter Biotech Technology SARL (Baxter International Inc.)
• Inventors: Anderson, David C., Mathews, Antony J., Stetler, Gary L.
• Primary Examiner(s): Guzo, David
• Assistant Examiner(s): SHUMAN, JON D
• Application Number:
  US09/058,562
• Time in Patent Office: 1,030 Days
• Field of Search: 530/385, 530/350, 530/402, 530/417, 530/829, 514/6, 514/832, 435/69.1, 435/69.6, 435/440, 435/320.1, 435/252.3, 435/254.11, 536/23.1, 536/23.5, 536/24.1
• US Class Current: 530/385
• 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: A61K 47/6445 Haemoglobin
• CPC Class Codes: C07K 14/805 Haemoglobins; Myoglobins
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/50 containing protease site
• CPC Class Codes: C07K 2319/92 containing an intein ("protein splicing")domain
• CPC Class Codes: C12N 15/81 for yeasts
• CPC Class Codes: Y10S 530/829 Blood

Abstract

DNA molecules which encode pseudodimeric globin-like polypeptides with an asymmetric cysteine mutation suitable for crosslinking two tetramers, or which encode pseudooligomeric globin-like polypeptides comprising four or more globin-like domains, are useful in the preparation of multimeric hemoglobin-like proteins.

Claims

1. A non-naturally occurring pseudotetrameric hemoglobin-like protein comprising a pseudodimeric polypeptide having two substantially homologous globin-like domains, one of which is mutated to provide an asymmetric crosslinkable cysteine residue, wherein the corresponding residue in the other globin-like domain of said pseudodimeric polypeptide is an amino acid other than cysteine, and wherein said pseudotetramer further comprises at least one other globin-like domain-bearing polypeptide, and wherein the asymmetric crosslinkable cysteine residue is a mutation in the alpha globin-like domain corresponding to a human alpha globin position selected from the group consisting of lys7, thr8, lys11, ala12, ,gly15, lys16, gly18, ala19, his20, glu23, and thr24.

2. The non-naturally occurring pseudotetrameric hemoglobin-like protein of claim 1 wherein the asymmetric crosslinkable cysteine residue is a mutation in the alpha globin-like domain corresponding to human alpha globin position lys16.

3. A non-naturally occurring pseudotetrameric hemoglobin-like protein comprising a pseudodimeric polypeptide having two substantially homologous globin-like domains, one of which is mutated to provide an asymmetric crosslinkable cysteine residue, wherein the corresponding residue in the other globin-like domain of said pseudodimeric polypeptide is an amino acid other than cysteine, and wherein said pseudotetramer further comprises at least one other globin-like domain-bearing polypeptide, and wherein the asymmetric crosslinkable cystine residue is a mutation in the beta globin-like domain corresponding to a human beta globin position selected from the group consisting of his2, leu3, thr4, glu6, ser9, thr12, ala13, gly16, lys17, val18, asn19, val20, asp21, and glu22.

4. The non-naturally occurring pseudotetrameric hemoglobin-like protein of claim 3 wherein the asymmetric crosslinkable cysteine residue is a mutation in the beta globin-like domain corresponding to human beta globin position ser9.

5. The non-naturally occurring pseudotetrameric hemoglobin-like protein of claim 3 wherein the asymmetric crosslinkable cysteine residue is a mutation in the beta globin-like domain corresponding to human beta globin position ala13.

6. A recombinant mutant hemoglobin-like protein comprising a mutation in a beta-globin like domain at amino acid position 93, wherein the cysteine at position 93 is replaced with an amino acid chosen from the group consisting of alanine and threonine.

7. The recombinant mutant hemoglobin-like protein of claim 6, wherein the cysteine at position 93 is replaced with alanine.