Expression of fully functional tetrameric human hemoglobin in Escherichia coli.
Synthetic 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 greater than 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 C4 reversed-phase HPLC profile essentially identical to that of human hemoglobin A0 and comigrates with hemoglobin A0 on SDS/PAGE. The visible spectrum and oxygen affinity are similar to that of native human hemoglobin A0. The recombinant protein shows a reduction in Bohr and phosphate effects, which may be attributed to the presence of methionine at the amino termini of the alpha and beta chains. We 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.
Hoffman
S J
SJ
Somatogen, Inc., Broomfield, CO 80021.
Looker
D L
DL
Roehrich
J M
JM
Cozart
P E
PE
Durfee
S L
SL
Tedesco
J L
JL
Stetler
G L
GL
D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D005813:Genes, Synthetic; D005914:Globins / Q000235:genetics*; D006454:Hemoglobins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000138:chemical synthesis; D009876:Operon; D010108:Oxyhemoglobins / Q000378:metabolism; D010833:Phytic Acid / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping
D006454:Hemoglobins; D046911:Macromolecular Substances; D015345:Oligonucleotide Probes; D010108:Oxyhemoglobins; D011994:Recombinant Proteins; D010833:Phytic Acid; D005914:Globins