A human recombinant haemoglobin designed for use as a blood substitute

Abstract

The need to develop a blood substitute is now urgent because of the increasing concern over blood-transmitted viral and bacterial pathogens. Cell-free haemoglobin solutions and human haemoglobin synthesized in Escherichia coli and Saccharomyces cerevisiae have been investigated as potential oxygen-carrying substitutes for red blood cells. But these haemoglobins cannot be used as a blood substitute because (1) the oxygen affinity in the absence of 2,3-bisphosphoglycerate is too high to allow unloading of enough oxygen in the tissues, and (2) they dissociate into alpha beta dimers that are cleared rapidly by renal filtration, which can result in long-term kidney damage. We have produced a human haemoglobin using an expression vector containing one gene encoding a mutant beta-globin with decreased oxygen affinity and one duplicated, tandemly fused alpha-globin gene. Fusion of the two alpha-globin subunits increases the half-life of this haemoglobin molecule in vivo by preventing its dissociation into alpha beta dimers and therefore also eliminates renal toxicity.

Development of Recombinant Hemoglobin-Based Oxygen Carriers

“…To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars in the erythrocytes (135). Expression of rHb in E. coli and S. cerevisiae was also accomplished with impressive yields (i.e., 2%-10% of the total cellular protein content consists of Hb) (2,28,52,62,74,79,96,97,136,156). Although each system has its own set of advantages and drawbacks, most recent efforts have focused on E. coli production systems.…”

“…Subsequently, Nagai developed a similar system for obtaining unfolded a-globin and reconstituting it with native bchains (92,125,146). Two years later, Somatogen's and Nagai's groups developed an Hb operon for coexpression of a-and bchains with the addition of exogenous heme to produce large amounts of intact, soluble tetrameric Hb in E. coli (62,79,80). Sligar's group developed a similar Hb operon system to express human adult human Hb A (HbA) (57,58).…”

“…Shen and C. Ho developed a system in which a-and bchains containing an extra N-terminal Met were coexpressed with E. coli methionine aminopeptidase to allow complete removal of the initiator amino acid and generation of recombinant Hb identical in a primary structure to native HbA (136,137). The Somatogen and Nagai groups also developed a fused di-a gene, which was inserted in an operon with a copy of the b-gene to express a tetramer that does not dissociate into a 1 b 1 dimers under normal physiological conditions, even when very dilute (79). The latter genetically crosslinked tetramer was called rHb0.1, with the 0.1 referring to one glycine linker between the two a-polypeptides, and this recombinant protein served as the genetic background for their initial rHBOC products.…”

Review of Anaesthesia and Analgesia in the Field With Emphasis on Airborne Military Operations

“…(e) Red cell substitutes: Presently investigations are still in progress on two artificial oxygen transporting fluids-stroma free haemoglobin (SFH) and perflurochemical emulsions (PFC) [22]. Once their clinical use starts, it will be remarkable progress for management of acute haemorrhagic shock.…”

Blood substitutes: refocusing an elusive goal

“…Scientists at Somatogen, in collaboration with the Medical Research Council of the UK, demonstrated expression in Escherichia coli after the introduction of the human genes into that organism (Hoffman et al, 1990). Somatogen subsequently designed a novel derivative, called`dialpha' haemoglobin that could also be expressed in E. coli (Looker et al, 1992). Dialpha haemoglobin is unique because the two a chains are fused, head-to-tail.…”