Expression of fully functional tetrameric human hemoglobin in Escherichia coli

Journal Article · Thu Nov 01 00:00:00 EST 1990 · Proceedings of the National Academy of Sciences of the United States of America; (United States)

DOI:https://doi.org/10.1073/pnas.87.21.8521· OSTI ID:5603314

Hoffman, S J; Looker, D L; Roehrich, J M; Cozart, P E; Durfee, S L; Tedesco, J L; Stetler, G L ^[1]

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1. Somatogen, Inc., Broomfield, CO (United States)

Synthesis genes encoding the human {alpha}- and {beta}-globin polypeptides have been expressed from a single operon in Escherichia coli. The {alpha}- and {beta}-globin polypeptides associate into soluble tetramers, incorporate heme, and accumulate to >5% of the total cellular protein. Purified recombinant hemoglobin has the correct stoichiometry of {alpha}- and {beta}-globin chains and contains a full complement of heme. Each globin chain also contains an additional methionine as an extension to the amino terminus. The recombinant hemoglobin has a C{sub 4} reversed-phase HPLC profile essentially identical to that of human hemoglobin A{sub 0} and comigrates with hemoglobin A{sub 0} on SDS/PAGE. The visible spectrum and oxygen affinity are similar to that of native human hemoglobin A{sub 0}. The authors have also expressed the {alpha}- and {beta}-globin genes separately and found that the expression of the {alpha}-globin gene alone results in a marked decrease in the accumulation of {alpha}-globin in the cell. Separate expression of the {beta}-globin gene results in high levels of insoluble {beta}-globin. These observations suggest that the presence of {alpha}- and {beta}-globin in the same cell stabilizes {alpha}-globin and aids the correct folding of {beta}-globin. This system provides a simple method for expressing large quantities of recombinant hemoglobin and allows facile manipulation of the genes encoding hemoglobin to produce functionally altered forms of this protein.

• OSTI ID: 5603314
• Journal Information: Proceedings of the National Academy of Sciences of the United States of America; (United States), Vol. 87:21; ISSN 0027-8424
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
BLOOD SUBSTITUTES
BIOCHEMISTRY
HEMOGLOBIN
GENE REGULATION
ESCHERICHIA COLI
GENE OPERONS
LIQUID COLUMN CHROMATOGRAPHY
OXYGEN
POLYPEPTIDES
PURIFICATION
BACTERIA
CARBOXYLIC ACIDS
CHEMISTRY
CHROMATOGRAPHY
DRUGS
ELEMENTS
GLOBINS
HEMATOLOGIC AGENTS
HETEROCYCLIC ACIDS
HETEROCYCLIC COMPOUNDS
MICROORGANISMS
NONMETALS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PEPTIDES
PIGMENTS
PORPHYRINS
PROTEINS
SEPARATION PROCESSES
550201* - Biochemistry- Tracer Techniques