Recombinant human hemoglobin: expression and refolding of beta-globin from Escherichia coli.
A plasmid analogous to the one described by Nagai and Thogersen (Nature, 309, 810-812, 1984) has been constructed for the expression of globins in E. coli. Induction with nalidixic acid produces high yields of a fusion protein, NS1-FX-beta-globin, where NS1 represents 81 residues of a flu virus protein and FX represents a blood-clotting Factor Xa recognition sequence, Ile-Glu-Gly-Arg. This fusion protein is readily solubilized in 50 mM NaOH and remains in solution when the pH is adjusted to 8.6. Under these conditions, the fusion protein is hydrolyzed by activated Factor X, giving authentic beta-globin which can be folded in the presence of cyanohemin and native alpha-chains to produce a tetrameric hemoglobin with the functional properties of natural human hemoglobin.
Fronticelli
C
C
Department of Biochemistry, University of Maryland, Medical School, Baltimore 21201.
O'Donnell
J K
JK
Brinigar
W S
WS
D000595:Amino Acid Sequence; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005170:Factor X / Q000378:metabolism; D015870:Gene Expression; D005914:Globins / Q000737:chemistry / Q000235:genetics*; D006454:Hemoglobins / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010100:Oxygen / Q000378:metabolism; D010957:Plasmids; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry; D013057:Spectrum Analysis
D006454:Hemoglobins; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D005170:Factor X; D005914:Globins; D010100:Oxygen