Somatogen, Inc.

Baxter Biotech Technology Sarl

Other

William Marsh Rice University

Baxter Biotech Technology Saarl

Antony James Mathews

David C Anderson

Gary L Stetler

John S Olson

Mary S Rosendahl

Douglas L Looker

Kiyoshi Nagai

Stephen J Hoffman

Michael Wagenbach

Spencer J Anthony-Cahill

Daniel H Doherty

Bruce A Kerwin

Christopher B Glascock

Douglas D Lemon

Stephen P Trimble

Janet K Epp

Kyoshi Nagai

Jacqueline F Aitken

Michael J Weickert

Peter O Olins

Maura-Ann H Matthews

James R Ryland

David W Traylor

David A Marquardt

Dominic G Madril

Steven L Durfee

Erin E Milne

J Jeffrey Plomer

Justinian O Neway

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.

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

The present invention relates to globins containing non-naturally occurring binding domains, In particular, the present invention is directed toward a binding domain comprising the oligomerizing domain GCN4 and GCN4 derivatives. Thc present invention also relates to multimeric hemoglobins comprised of at least one globin containing at least one non-naturally occurring binding domain.

Globins containing binding domains

This invention relates to recombinant hemoglobins containing mutations in or near the heme pocket of the hemoglobin molecule. This invention particularly relates to recombinant hemoglobins that have altered geometry or polarity around the distal portion of the heme pocket.

Nucleic acids encoding mutant recombinant hemoglobins containing heme pocket mutations

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.

Production and use of multimeric hemoglobins

Modified hemoglobin-like compounds and methods of purifying same

Mutant recombinant hemoglobins containing heme pocket mutations

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.

Genetically fused globin-like polypeptides having hemoglobin-like

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. We further describe the production of tetrameric human hemoglobin and di-alpha/beta.sub.2 hemoglobin in the yeast Saccharomyces cerevisiae. The synthesis of the protein is directed by a synthetic promotor consisting of two functional parts, an upstream activator sequence (UAS) that confers inducible transcription by galactose from a consensus yeast transcriptional initiation site. The expression construct is designed such that translation is expected to initiate at the same position as the human wild-type genes for .alpha.- and .beta.-globin. Three different types of expression vectors have been used: (1) .alpha.-globin and .beta.-globin contained on two separate plasmids (pGS4688 and pGS4988) in a diploid yeast strain; (2) .alpha.-globin and .beta.-globin each contained on a single plasmid (pGS289 and pGS389) and expressed in either haploid or diploid strains; and (3) di-alpha-globin and beta globin contained on a single plasmid (pGS 3089)and expressed in haploid strains. Finally, we describe the co-expression of alpha and beta globin chains. The chains are folded together and combined intracellularly with heme to form active tetrameric hemoglobin. The hemoglobin may be recovered from the cells' soluble fraction. The invention thus obviates the need to express alpha and beta globin separately, solubilize, renature and purify them, and combine them in vitro with heme to obtain an artificial hemoglobin. By way of comparison, the separately expressed beta globin known in the art is deposited in inclusion bodies. Polycistronic co-expression of alpha and beta globins is particularly preferred.

Hemoglobin-like protein comprising genetically fused globin-like

The present invention relates to modified hemoglobin-like compounds. The novel compounds include a globin-like polypeptide containing at least two di-alpha domains and multimeric hemoglobin-like proteins having a core hemoglobin-like moiety directly attached to at least two other hemoglobin-like moieties. The invention also relates to nucleic acid molecules encoding the novel polypeptides. Methods of making the multimeric hemoglobin-like proteins are also provided, as well as compositions containing the proteins.

Modified hemoglobin-like compounds

The present invention generally relates to methods for purifying hemoglobin solutions and to hemoglobin solutions obtained by the methods. In one aspect, such methods include removing contaminants in crude hemoglobin-containing lysates with heat treatment. In a further aspect, the present invention provides methods for producing substantially purified hemoglobin solutions using immobilized metal affinity chromatography, optionally following by anion exchange chromatography.

Purification of hemoglobin

DNA encoding fused alpha-beta globin pseuodimer and production of

A tracer, such as a radioisotope, is conjugated to a hemoglobin-like protein, such as normal hemoglobin, for use as an in vitro diagnostic imaging agent.

Hemoglobins as drug delivery agents

DNA encoding fused di-beta globins and production of pseudotetrameric

DNA for the production of multimeric hemoglobins

Growing community of inventors

Louisville, CO, United States of America

Antony James Mathews