Turnover of recombinant human hemoglobin in Escherichia coli occurs rapidly for insoluble and slowly for soluble globin.
Co-expression of di-alpha-globin and beta-globin in Escherichia coli in the presence of exogenous heme yielded high levels of soluble, functional recombinant human hemoglobin (rHb1.1) and, under certain conditions, large amounts of insoluble globin protein. Insoluble rHb1.1 accumulated in large, amorphous inclusion bodies visible by electron microscopy. The half-life of soluble rHb1.1 in E. coli, measured by pulse-chase experiments, was at least 11 h for each globin subunit. The in vivo half-life for insoluble globin was about fivefold shorter than that for soluble rHb1.1. We expressed significant amounts of each subunit, di-alpha-globin and beta-globin, independently with exogenous heme. The half-life of the soluble subunits alone was approximately 1 and 4 h, respectively, shorter than when they were expressed together as rHb1.1. Individually, the insoluble di-alpha-globin subunit had a half-life of just under 1 h when exogenous heme was added, but under 20 min when exogenous heme was not provided. The greater persistence of insoluble subunits in the presence of heme indicated that heme may stabilize the insoluble globin protein. The soluble rHb1.1 persistence in the E. coli cytoplasm during long periods of stationary phase growth indicated that once assembled, rHb1.1 is extremely resistant to proteolysis.
Weickert
M J
MJ
Somatogen, Inc., Boulder, Colorado 80301, USA. Weickert@aol.com
Curry
S R
SR
D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D015870:Gene Expression / Q000235:genetics; D005914:Globins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D006454:Hemoglobins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D002479:Inclusion Bodies / Q000648:ultrastructure; D007700:Kinetics; D008854:Microscopy, Electron; D011233:Precipitin Tests; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012995:Solubility; D013462:Sulfur Radioisotopes / Q000378:metabolism
D006454:Hemoglobins; D011994:Recombinant Proteins; D013462:Sulfur Radioisotopes; D006418:Heme; D005914:Globins; D010450:Endopeptidases