Enhancing Stability and Expression of Recombinant Human Hemoglobin in E. Coli: Progress in the Development of a Recombinant HBOC Source

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dc.contributor.advisorOlson, John S.en_US
dc.contributor.committeeMemberMacKenzie, Kevinen_US
dc.contributor.committeeMemberTao, Yitzi Janeen_US
dc.contributor.committeeMemberWagner, Danielen_US
dc.creatorGraves, Philip Edwaren_US
dc.date.accessioned2018-12-18T21:35:15Zen_US
dc.date.available2018-12-18T21:35:15Zen_US
dc.date.issued2008en_US
dc.description.abstractThe commercial feasibility of recombinant human Hb (rHb) as an O2 delivery pharmaceutical is limited by the production yield of holoprotein in E. coli. Currently the production ofrHb 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° 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. aGlyl5 to Ala and P Glyl6 to Ala mutations have been constructed to increase the stability of the A helices of both subunits of adult human hemoglobin (HbA), based on comparison with the sequences of the more stable sperm whale hemoglobin subunits. Human fetal hemoglobin is also known to be more stable than HbA, and comparisons between human P and y (fetal Hb) chains indicate several substitutions that stabilize the aiPi interface, one of which, pHisllb to lie, 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 may enhance holohemoglobin production to levels that may make rHb a plausible starting material for all extracellular Hb-based oxygen carriers.en_US
dc.format.digitalOriginreformatted digitalen_US
dc.format.extent75 ppen_US
dc.identifier.callnono call numberen_US
dc.identifier.citationGraves, Philip Edwar. "Enhancing Stability and Expression of Recombinant Human Hemoglobin in E. Coli: Progress in the Development of a Recombinant HBOC Source." (2008) Master’s Thesis, Rice University. <a href="https://hdl.handle.net/1911/104917">https://hdl.handle.net/1911/104917</a>.en_US
dc.identifier.digitalRICE2569en_US
dc.identifier.urihttps://hdl.handle.net/1911/104917en_US
dc.language.isoengen_US
dc.rightsCopyright is held by the author, unless otherwise indicated. Permission to reuse, publish, or reproduce the work beyond the bounds of fair use or other exemptions to copyright law must be obtained from the copyright holder.en_US
dc.titleEnhancing Stability and Expression of Recombinant Human Hemoglobin in E. Coli: Progress in the Development of a Recombinant HBOC Sourceen_US
dc.typeThesisen_US
dc.type.materialTexten_US
thesis.degree.departmentBiochemistry and Cell Biologyen_US
thesis.degree.disciplineNatural Sciencesen_US
thesis.degree.grantorRice Universityen_US
thesis.degree.levelMastersen_US
thesis.degree.nameMaster of Artsen_US
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