Somatogen, Inc.

Baxter Biotech Technology Sarl

Douglas L. Looker

Stephen J. Hoffman

Rhb1 Acquisition Corp.

Michael Wagenbach

Mary S. Rosendal

Gary L. Stetler

Kiyoshi Nagai

Gary L Stetler

David C Anderson

Antony James Mathews

Douglas L Looker

Stephen J Hoffman

Mary S Rosendahl

Spencer J Anthony-Cahill

Maura-Ann H Matthews

Mary S Rosendal

A functional linker for a polypeptide in which two alpha or beta globin-like domains are genetically fused is determined by screening a library of genetically fused polypeptides, in which the linker region is varied, for the ability to participate in the formation of hemoglobin-like protein, as measured by the protein's response to carbon monoxide. In a preferred embodiment, cells expressing the protein turn red as a result of carbon monoxide pressure.

Method of determining a functional linker for fusing globin subunts

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

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

Co-expression of alpha and beta globins

DNA encoding fused di-beta globins and production of pseudotetrameric

DNA for the production of multimeric hemoglobins

The present invention relates to regulators of cellular gene transcription, particularly inhibitors of cellular gene transactivating factors and in particular to inhibition of gene transcription in a viral host cell that is subject to regulation by proteins or factors that originate from a virus as well as conjugates or fusion products of the inhibitors and internalization molecules, pharmaceutical compositions that can be used to alleviate or prevent the manifestation of disease states that are the result of unregulated DNA transcription as a result of transactivation, methods of treating diseases that are caused or exacerbated by the presence of transactivating factors, and regulated gene therapy to achieve long term drug delivery of the inhibitors of the present invention. This invention can be applied both to cells with genetic abnormalities or to cells infected with a virus. Preferably, at least one protein of the protein-protein interactions is a transactivating factor.

Peptide inhibitors of tax-dependent transcription

Growing community of inventors

Denver, CO, United States of America

Gary L Stetler