DNA encoding fused di-alpha globins and use thereof

• US 6,828,125 B1
• Filed: 05/30/1995
• Issued: 12/07/2004
• 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: Nagai, Kiyoshi, Looker, Douglas L., Hoffman, Stephen J.
• Primary Examiner(s): CARLSON, KAREN C
• Application Number:
  US08/453,485
• Time in Patent Office: 3,479 Days
• Field of Search: 530/385, 536/23.1, 435/69.1, 435/320.1, 435/240.1, 435/69.6
• US Class Current: 435/69.6
• 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: C12N 15/81 for yeasts

Abstract

The alpha subunits of hemoglobin, which in nature are formed as separate polypeptide chains which bind noncovalently to the beta subunits, are here provided in the form of the novel molecule di-alpha globin, a single polypeptide chain defined by connecting the two alpha subunits either directly via peptide bond or indirectly by a flexible amino acid or peptide linker. Di-alpha globin may be combined in vivo or in vitro with beta globin and heme to form hemoglobin. Di-alpha globin is expressed by recombinant DNA techniques. Di-beta globin may be similarly obtained.

Claims

1. A DNA molecule comprising a first DNA sequence encoding a non-naturally occurring genetically fused pseudooligomeric polypeptide comprising two or more alpha globin-like domains connected by one or more peptide bonds into a single unbranched polypeptide chain, said chain being capable of associating with beta globin and incorporating heme to form a hemoglobin-like protein.

2. The DNA molecule of claim 1 which comprises only two globin-like domains.

3. The DNA molecule of claim 1 wherein a first and a second globin-like polypeptide domain are connected by a peptide linker of one or more amino acids.

4. The DNA molecule of claim 3 wherein the peptide linker is not more than nine amino acids.

5. The DNA molecule of claim 3 wherein the amino acids of the linker are selected from the group consisting of lysine, aspartic acid, arginine, serine, asparagine, proline and glycine.

6. The DNA molecule of claim 5 wherein the linker consists of one or more glycines.

7. The DNA molecule of claim 5 wherein the amino acids of the linker are selected from the group consisting of glycine and proline.

8. The DNA molecule of claim 1 wherein each globin-like domain has at least 75% sequence identity with human alpha globin.

9. The DNA molecule of claim 1 wherein each globin-like domain is essentially identical in sequence with human alpha globin.

10. The DNA molecule of claim 1 wherein said polypeptide comprises only two globin-like domains.

11. The DNA molecule of claim 1 wherein said globin-like domains are essentially identical, but wherein at a predetermined position, a first globin-like domain has a cysteine residue and a second globin-like domain has a residue other than cysteine.

12. The DNA molecule of claim 1 wherein said protein is a low affinity hemoglobin mutant.

13. The DNA molecule of claim 1 wherein said protein is a high affinity hemoglobin mutant.

14. The recombinant DNA molecule of claim 1 further comprising a second DNA sequence encoding a beta globin-like polypeptide.

15. The DNA molecule of claim 14 wherein the beta globin-like polypeptide comprises a Presbyterian mutation.

16. The molecule of claim 14, wherein the DNA sequence encoding the pseudooligomeric polypeptide defines a single cistron of a polycistronic operon, and the second DNA sequence which defines another cistron of the polycistronic operon, wherein the cistrons are transcribed from a common promoter.

17. The molecule of claim 16 wherein the common promoter is the Tac promoter.

18. The DNA molecule of claim 1 wherein the linker comprises one more glycines.

19. The DNA molecule of claim 1 wherein each globin-like domain is essentially identical in sequence with a vertebrate alpha globin.

20. The DNA molecule of claim 1 wherein each globin-like domain is essentially identical in sequence with a mammalian alpha globin.

21. The DNA molecule of claim 1 wherein each globin-like domain comprises, at corresponding positions, the amino acids residues which are invariant among vertebrate alpha globins.

22. The DNA molecule claim 1 wherein each globin-like domain comprises, at corresponding positions, the amino acid residues which are invariant among mammalian alpha globins.

23. The DNA molecule of claim 1 wherein each alpha globin-like domain differs from at least one vertebrate alpha globin solely by mutations corresponding to an allelic variation in human alpha globin and/or substitutions wherein each substituted amino acid is replaced by another in the same exchange group, the exchange groups being:

I Ala, Ser, Thr, Pro, Gly

II Asn, Asp, Glu, Gln

III His, Arg, Lys

IV Met, Leu, Ile, Val, Cys

V Phe, Tyr, Trp.

24. The DNA molecule of claim 1 wherein each alpha globin-like domain differs from at least one mammalian alpha globin solely by mutations corresponding to an allelic variation in human alpha globin and/or substitutions wherein each substituted amino acid is replaced by another in the same exchange group, the exchange groups being:

I Ala, Ser, Thr, Pro, Gly

II Asn, Asp, Glu, Gln

III His, Arg, Lys

IV Met, Leu, Ile, Val, Cys

V Phe, Thy, Trp.

25. The DNA molecule of claim 1 wherein each alpha globin-like domain differs from at least one human alpha globin solely by mutations corresponding to an allelic variation in human alpha globin and/or substitutions wherein each substituted amino acid is replaced by another in the same exchange group, the exchange groups being:

I Ala, Ser, Thr, Pro, Gly

II Asn, Asp, Glu, Gln

III His, Arg, Lys

IV Met, Leu, Ile, Val, Cys

V Phe, Tyr, Trp.

26. The DNA molecule of claim 1 wherein each alpha globin-like domain has at least 75% sequence identity with a mammalian alpha globin.

27. The DNA molecule of claim 1 wherein each alpha globin-like domain has at least the same degree of sequence identity with human alpha globin as does the corresponding chicken globin.

28. The DNA molecule of claim 1 wherein each alpha globin-like domain has at least the same degree of sequence identity with human alpha globin as does the corresponding carp globin.

29. The DNA molecule of claim 1 wherein each alpha globin-like domain has at least the same degree of sequence identity with human alpha globin as does the corresponding platypus globin.

30. The DNA molecule of claim 1 wherein the linker comprises two or more glycines, and at least the first and last amino acids of the liner are glycines.

31. The DNA molecule of claim 30 wherein the amino acids of the linker are selected from the group consisting of alanine, threonine, lysine, aspartic acid, arginine, serine, asparagine, proline and glycine.

32. The DNA molecule of claim 1 wherein said pseudooligomeric polypeptide comprises two or more pairs of alpha globin-like domains, and is capable of associating with one or more beta globin-like polypeptides to form a polytetrameric hemoglobin-like protein with reversible oxygen binding activity.

33. The DNA molecule of claim 32 wherein the polypeptide further comprises a peptide linker between at least one pair of successive alpha globin-like domains is at least 17 Å

in length.

34. The DNA molecule of claim 32 wherein the polypeptide further comprises a peptide linker between at least one pair of successive alpha globin-like domains is at least 22 Å

in length.

35. The DNA molecule of claim 32 wherein said polypeptide genetically fuses two or more di-alpha globin-like polypeptides.

36. The DNA molecule of claim 1 wherein said hemoglobin-like protein is a low oxygen affinity mutant hemoglobin.

37. The DNA molecule of claim 36 wherein at least one of said low affinity mutations is a mutation of an alpha-globin mutation selected from the group consisting of 43 phe→

leu, 43 phe→

val, 86 leu→

arg, 94 asp→

asn, 46 phe→

thr, 46 phe→

ser, 46 phe→

ala, 58 his→

phe, 58 his→

trp, 61 lys→

thr, 61 lys→

ser, 61 lys→

met, 61 lys→

asn, 62 val→

leu, 62 val→

ile, 62 val→

phe, 62 val→

trp, 65 ala→

asp, 94 asp→

gln, 94 asp→

thr, 94 asp→

ser, 94 asp→

lys, 94 asp→

gly, and 94 asp→

arg.

38. The DNA molecule of claim 1 wherein said hemoglobin-like protein is a high oxygen affinity mutant hemoglobin.

39. The DNA molecule of claim 38 wherein at least one of said high affinity mutations is a mutation of an alpha globin-like domain which corresponds to a human alpha globin mutation selected from the group consisting of 6 Asp→

Ala, 6 Asp→

Asn, 6 Asp→

Val, 6 Asp→

Tyr, 6 Lys→

Asn, 40 Lys→

Glu, 44 Pro→

Leu, 44 Pro→

Arg, 45 His→

Arg, 85 Asp→

Asn, 92 Arg→

Gln, 92 Arg→

Leu, 95 Pro→

Leu, 95 Pro→

Ser, 95 Pro→

Ala, 95 Pro→

Arg, 97 Asn→

Lys, 126 Asp→

Asn, 141 Arg→

His, 141 Arg→

Ser, and 141 Arg→

Leu.

40. The DNA molecule of claim 1 wherein at least one alpha globin-like domain has a cysteine at a position which is not a cysteine in human alpha globin.

41. The DNA molecule of claim 1 wherein cysteine is at a position in said domain corresponding to a position in the alpha globin-like domain corresponding to a human alpha globin position selected from the group consisting of his72, asn78, asn68, ala71, thr67, lys7, lys11, thr8, ala12, thr118, lys16, ala45, glu116, gly15, his112, thr24, glu23, lys60, lys56, his50, gly51, glu53, ser49, asp47, gln54, his45, lys90, ala82, lys61, ala19, his20, asp85, ser81, asp75, asp74, lys139, asp64, and gly18.

42. The DNA molecule of claim 1 wherein each alpha globin-like domain of said genetically fused polypeptide is identical to human alpha globin save for

(a) one or more an oxygen affinity-modifying modifications characterized as

(i) one or more low affinity mutations, selected from the group consisting of 43 phe→

leu, 43 phe→

val, 86 leu→

arg, 94 asp→

asn, 46 phe→

thr, 46 phe→

ser, 46 phe→

ala, 58 his→

phe, 58 his→

trp, 61 lys→

thr, 61 lys→

ser, 61 lys→

met, 61 lys→

asn, 62 val→

leu, 62 val→

ile, 62 val→

phe, 62 val→

trp, 65 ala→

asp, 94 asp→

gln, 94 asp→

thr, 94 asp→

ser, 94 asp→

lys, 94 asp→

gly, and 94 asp→

arg, or

(ii) one or more high affinity mutations selected from the group consisting of 6 Asp→

Ala, 6 Asp→

Asn, 6 Asp→

Val, 6 Asp→

Tyr, 6 Lys→

Asn, 40 Lys→

Glu, 44 Pro→

Leu, 44 Pro→

Arg, 45 His→

Arg, 85 Asp→

Asn, 92 Arg→

Gln, 92 Arg→

Leu, 95 Pro→

Leu, 95 Pro→

Ser, 95 Pro→

Ala, 95 Pro→

Arg, 97 Asn→

Lys, 126 Asp→

Asn, 141 Arg→

His, 141 Arg→

Ser, and 141 Arg→

Leu;

and/or

(b) a Cys substitution at one or more sites selected from the group consisting of;

his72, asn78, asn68, ala71, thr67, lys7, lys11, thr8, ala12, thr118, lys16, ala45, glu16, gly15, his112, thr24, glu23, lys60, lys56, his50, gly51, glu53, ser49, asp47, gln54, his45, lys90, ala82, lys61, ala19, his20, asp85, ser81, asp75, asp74, lys139, asp64, and gly18.

43. A method of producing a human hemoglobin-like protein with reversible oxygen binding activity which comprises providing a host transformed with a recombinant DNA molecule according to claim 1, cultivating said host under conditions whereunder it expresses said pseudooligomeric polypeptide, and combining said polypeptide with beta globin and heme to obtain a human hemoglobin like protein.

44. The method of claim 43 wherein the host is a yeast cell.

45. The method of claim 43 wherein the host is a bacterial cell.

46. A method of producing a human hemoglobin-like protein with reversible oxygen binding activity which comprises providing a host transformed with a recombinant DNA molecule according to claim 16, cultivating the host under conditions where under it expresses the pseudooligomeric polypeptide and the beta globin-like polypeptide, wherein the di-alpha globin pseudooligomeric polypeptide and the beta-globin-like polypeptide, are co-expressed from the recombinant DNA molecule and heme is provided by said host and incorporated to form said hemoglobin-like protein.