title: Enhancing stability and expression of recombinant human hemoglobin in E. coli: Progress in the development of a recombinant HBOC source.
abstract: The commercial feasibility of recombinant human Hb (rHb) as an O(2) delivery pharmaceutical is limited by the production yield of holoprotein in E. coli. Currently the production of rHb is not cost effective for use as a source in the development of third and fourth generation Hb-based oxygen carriers (HBOCs). The major problems appear to be aggregation and degradation of apoglobin at the nominal expression temperatures, 28-37 degrees C, and the limited amount of free heme that is available for holohemoglobin assembly. One approach to solve the first problem is to inhibit apoglobin precipitation by a comparative mutagenesis strategy to improve apoglobin stability. alpha Gly15 to Ala and beta Gly16 to Ala mutations have been constructed to increase the stability of the alpha helices of both subunits of HbA, based on comparison with the sequences of the more stable sperm whale hemoglobin subunits. Fetal hemoglobin is also known to be more stable than human HbA, and sequence comparisons between human beta and gamma (fetal Hb) chains indicate several substitutions that stabilize the alpha1beta1 interface, one of which, beta His116 to Ile, increases resistance to denaturation and enhances expression in E. coli. These favorable effects of enhanced globin stability can be augmented by co-expression of bacterial membrane heme transport systems to increase the rate and extent of heme uptake through the bacterial cell membranes. The combination of increased apoglobin stability and active heme transport appear to enhance holohemoglobin production to levels that may make rHb a plausible starting material for all extracellular Hb-based oxygen carriers.
authors.affiliation: Department of Biochemistry and Cell Biology, Rice University, 6100 N. Main, Houston, TX 77005, USA. pgraves@rice.edu
authors.forename: Philip E
authors.initials: PE
authors.lastname: Graves
authors.forename: Douglas P
authors.initials: DP
authors.lastname: Henderson
authors.forename: Molly J
authors.initials: MJ
authors.lastname: Horstman
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Solomon
authors.forename: John S
authors.initials: JS
authors.lastname: Olson
mesh_terms: D001802:Blood Substitutes / Q000737:chemistry*; D003001:Cloning, Molecular; D003362:Cost-Benefit Analysis; D004355:Drug Stability; D004926:Escherichia coli / Q000235:genetics; D005914:Globins / Q000737:chemistry / Q000235:genetics; D006418:Heme / Q000378:metabolism; D006454:Hemoglobins / Q000737:chemistry* / Q000191:economics / Q000235:genetics*; D006801:Humans; D015202:Protein Engineering / Q000191:economics / Q000379:methods*; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry* / Q000191:economics
chemical_list: D001802:Blood Substitutes; D006454:Hemoglobins; D011994:Recombinant Proteins; D006418:Heme; D005914:Globins