Hemoglobin mutants with increased soluble expression and/or reduced nitric oxide scavenging

• US 7,049,406 B2
• Filed: 03/27/2002
• Issued: 05/23/2006
• Est. Priority Date: 05/02/1997
• Status: Expired due to Fees

• Current Assignee: Baxter Biotech Technology SARL (Baxter International Inc.), Rice University
• Original Assignee: Baxter Biotech Technology SARL (Baxter International Inc.), Rice University
• Inventors: Lemon, Douglas D, Mathews, Antony J, Doherty, Daniel H, Weickert, Michael J, Glascock, Christopher B, Olson, John S
• Primary Examiner(s): CARLSON, KAREN C
• Application Number:
  US10/107,871
• Publication Number:
  US 20030017537A1
• Time in Patent Office: 1,518 Days
• Field of Search: 530/385
• 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: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/06 Antianaemics
• CPC Class Codes: A61P 7/08 Plasma substitutes; Perfusion solutions; Dialytics or haemodialytics; Drugs for electrolytic or acid-base disorders, e.g. hypovolemic shock artificial tears A61P27/04
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/08 Vasodilators for multiple indications
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C07K 14/805 Haemoglobins; Myoglobins

Abstract

The invention relates to novel recombinant hemoglobins having reduced nitric oxide scavenging and/or increased high soluble expression. The invention further relates to methods of increasing the soluble expression of recombinant hemoglobin by adding exogenous hemin in molar excess of the heme binding sites of recombinant hemoglobin.

Claims

1. A method of obtaining high expression of soluble recombinant hemoglobin, comprising:

(a) incorporating into providing a host cell a vector containing a gene nucleotide sequences encoding globins capable of associating to form a functional hemoglobin, wherein the nucleotide sequence encoding a β

globin having at least one mutation or combination of mutations that directs high soluble expression of the recombinant hemoglobin selected from the group consisting of;

D73Y;

D73I;

D73M;

D73E;

D73T;

D73K;

D73L;

K82D;

K82E;

K82G;

K82P;

K82Q;

K82S;

K82N;

K82H;

N102A;

N102V;

K82D(Providence)+D73G(Tilburg);

K82D(Providence)+D73Y(Vancouver);

V67W+D73E;

V67W+D73I;

V67W+D73Y(Vancouver);

V67W+D73E+K82D;

(Providence)

V67W+D73I+K82D;

(Providence)

V67W+D73Y(Vancouver)+K82D;

(Providence);

V67W+K82E; and

V67W+K82G

(b) inducing the host cell to express soluble recombinant hemoglobin; and

(c) purifying the soluble recombinant hemoglobin.

2. The method of claim 1, further comprising adding excess hemin in step (b).

3. The method of claim 1, wherein said mutation is selected from the group consisting of D73I, D73M, D73E, D73T, D73Y, K82D, K82E, K82G, K82P, K82Q, K82S, K82N, N102A and N102V.

6. The method of claims 2 or 4, wherein at least a 3-fold molar excess of heme is added.

4. A method of obtaining high expression of soluble recombinant hemoglobin wherein at least one β

globin of the recombinant hemoglobin has at least one mutation or combination of mutations selected from the group consisting of D73Y, D73I, D73M, D73E, D73T, D73K, D73L, K82D, K82E, K82G, K82P, K82Q, K82S, K82N, N102A, N102V, K82D(Providence)+D73G(Tilburg), K82D(Providence)+D73Y (Vancouver), V67W+D73E, V67W+D73I, V67W+D73Y (Vancouver), V67W+D73E+K82D V67W+D73I+K82D(Providence), V67W+D73Y (Vancouver)+K82D(Providence), V67W+K82E, and V67W+K82G, comprising;

(a) adding excess hemin to a culture of a host cell capable of expressing the soluble recombinant hemoglobin;

(b) inducing the host cell to express the soluble recombinant hemoglobin; and

(c) purifying the soluble recombinant hemoglobin.

5. The method of claim 4, wherein the recombinant hemoglobin comprises at least one beta globin mutation or a combination of beta globin mutations selected from the group consisting of:

D73Y;

D73I;

D73M;

D73E;

D73T;

K82D;

K82E;

K82G;

K82P;

K82Q;

K82S;

K82N;

N102A; and

N102V.