title: Stabilization of apoglobin by low temperature increases yield of soluble recombinant hemoglobin in Escherichia coli.
abstract: Accumulation of soluble recombinant hemoglobin (rHb1.1) in Escherichia coli requires proper protein folding, prosthetic group (heme) addition, and subunit assembly. This served as a new model system for the study of the effects of temperature, protein synthesis rates, and protein accumulation rates on protein solubility in E. coli. Fermentation expression of rHb1.1 at 30 degrees C from cultures containing a medium or high globin gene dosage (pBR-based or pUC-based plasmids with rHb1.1 genes under the control of the tac promoter) was compared. A medium gene dosage resulted in rHb1.1 accumulating to approximately 7% of the soluble cell protein, of which 78% was soluble. A high globin gene dosage resulted in a > or = 3-fold increase in total globin to 23 to 24% of the soluble cell protein, but 70% was insoluble. Accumulation of insoluble rHb1.1 began immediately upon induction. The proportion of rHb1.1 from the high globin gene dosage that accumulated as insoluble globin was affected by reducing (i) the inducer concentration and (ii) the temperature. Reducing the inducer concentration reduced globin synthesis up to eightfold but increased the proportion of soluble rHb1.1 to 93%. In contrast, total globin protein synthesis was barely affected by reducing the temperature from 30 to 26 degrees C, while soluble globin accumulation increased > 2-fold to approximately 15% of the soluble cell protein. The contrast between the effects of reducing rates of protein synthesis and accumulation and those of reducing temperature suggests that lower temperature stabilizes one or more folding intermediates. We propose a simplified physical model which integrates protein synthesis, folding, and heme association. This model shows that temperature-dependent apoglobin stability is the most critical factor in soluble rHb1.1 accumulation.
authors.affiliation: Somatogen, Inc., Boulder, Colorado 80301, USA. Weickert@AOL.COM
authors.forename: M J
authors.initials: MJ
authors.lastname: Weickert
authors.forename: M
authors.initials: M
authors.lastname: Pagratis
authors.forename: S R
authors.initials: SR
authors.lastname: Curry
authors.forename: R
authors.initials: R
authors.lastname: Blackmore
mesh_terms: D001059:Apoproteins / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D005285:Fermentation; D018628:Gene Dosage; D006454:Hemoglobins / Q000096:biosynthesis* / Q000378:metabolism*; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis*; D013696:Temperature
chemical_list: D001059:Apoproteins; D006454:Hemoglobins; D011994:Recombinant Proteins; C023478:apohemoglobin