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|Title:||Total gene synthesis, bacterial expression and functional characterization of a recombinant human hemoglobin: Progress towards a blood substitute|
|Author(s):||Hernan, Ronald Allen|
|Doctoral Committee Chair(s):||Sligar, Stephen G.|
|Department / Program:||Biochemistry|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||To better understand the role of specific residues of hemoglobin in allosteric function, cooperative interactions, ligand discrimination and protein-protein recognition, a genetic handle is required. The use of total gene synthesis alleviates some of the laborious procedures associated with conventional cDNA cloning and circumvents the problems associated with the expression of human hemoglobin chains from cDNA clones. Cloning of individual alpha and beta genes in pUC18 failed to express either gene. However construction of a hemoglobin operon with beta chain down stream of alpha chain inserted into pUC18 over-expressed human hemoglobin. The two proteins combine intracellularly with endogenous heme which is concomitantly overproduced to yield tetrameric hemoglobin as toughly 5-10% of total E. coli proteins.
Physical characterization of the purified recombinant hemoglobin indicate that the over expressed protein is identical to native hemoglobin. Functional characterization of the recombinant hemoglobin, however, reveals significant differences when compared to native hemoglobin. These differences include a reduction in cooperativity as measured by the hill coefficient, and a loss of interaction with heterotropic effectors 2,3,-diphosphoglycerate and protons. In addition CO combination kinetic analysis of recombinant hemoglobin revealed a heterogeneous population of high affinity active sites. These alterations in reactivity indicate that the recombinant hemoglobin expressed in E. coli has altered deoxy structure.
Separation and characterization of isolated recombinant alpha and beta chains indicate that they are functionally identical to native chains. Reassembly of the recombinant hemoglobin tetramer form isolated chains resulted in a protein which had cooperativity similar to native hemoglobin and had identical response to allosteric effectors 2,3-diphosphoglycerate, inositolhexaphosphate, and protons. Subsequently, CO combination kinetics of the reassembled tetramer demonstrated a homogeneous population of heme reactivates which respond to allosteric effectors. The methods described within this thesis suggest alterations occur during in vivo assembly of the recombinant hemoglobin. However these perturbations can be overcome by reassembling the recombinant tetramer for isolated chains. This reassembly allows a vehicle by which mutagenesis studies coupled with detailed analysis can be used to understand structure-function relationships.
|Rights Information:||Copyright 1994 Hernan, Ronald Allen|
|Date Available in IDEALS:||2011-05-07|
|Identifier in Online Catalog:||AAI9503212|
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