Expression and Purification of Recombinant Hemoglobin in Escherichia coli

Natarajan, Chandrasekhar; Jiang, Xiaoben; Fago, Angela; Weber, Roy E.; Moriyama, Hideaki; Storz, Jay F.

doi:10.1371/journal.pone.0020176

Cited by 51 publications

(64 citation statements)

References 40 publications

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“…The twolocus HBB haplotype that increases in frequency as a function of altitude (d 1 d 1 ) is associated with increased hemoglobinoxygen affinity (Storz et al , 2010aNatarajan et al 2011), which enhances pulmonary oxygen loading under hypoxia. Thus, the deer mouse b-globin polymorphism represents a rare case in which the spatial patterning of allele frequency variation across an environmental gradient can be related to adaptive functional properties of the encoded protein.…”

Section: Discussionmentioning

confidence: 99%

Altitudinal Variation at Duplicated β-Globin Genes in Deer Mice: Effects of Selection, Recombination, and Gene Conversion

et al. 2012

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Spatially varying selection on a given polymorphism is expected to produce a localized peak in the between-population component of nucleotide diversity, and theory suggests that the chromosomal extent of elevated differentiation may be enhanced in cases where tandemly linked genes contribute to fitness variation. An intriguing example is provided by the tandemly duplicated b-globin genes of deer mice (Peromyscus maniculatus), which contribute to adaptive differentiation in blood-oxygen affinity between high-and low-altitude populations. Remarkably, the two b-globin genes segregate the same pair of functionally distinct alleles due to a history of interparalog gene conversion and alleles of the same functional type are in perfect coupling-phase linkage disequilibrium (LD). Here we report a multilocus analysis of nucleotide polymorphism and LD in highland and lowland mice with different genetic backgrounds at the b-globin genes. The analysis of haplotype structure revealed a paradoxical pattern whereby perfect LD between the two b-globin paralogs (which are separated by 16.2 kb) is maintained in spite of the fact that LD within both paralogs decays to background levels over physical distances of less than 1 kb. The survey of nucleotide polymorphism revealed that elevated levels of altitudinal differentiation at each of the b-globin genes drop away quite rapidly in the external flanking regions (upstream of the 59 paralog and downstream of the 39 paralog), but the level of differentiation remains unexpectedly high across the intergenic region. Observed patterns of diversity and haplotype structure are difficult to reconcile with expectations of a two-locus selection model with multiplicative fitness.

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Section: Discussionmentioning

confidence: 99%

Altitudinal Variation at Duplicated β-Globin Genes in Deer Mice: Effects of Selection, Recombination, and Gene Conversion

et al. 2012

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“…To produce rHbs for the protein engineering experiments, the α A -and β A -globin genes of A. melanogenys were synthesized by Genscript after optimizing nucleotide sequences with respect to Escherichia coli codon preferences. Gene cassettes for the α A -and β A -globin genes and the methionine aminopeptidase (MAP) gene were tandemly cloned into the custom pGM expression plasmid described by Natarajan et al (21). All rHbs were expressed in the JM109 (DE3) E. coli strain.…”

Section: Methodsmentioning

confidence: 99%

Repeated elevational transitions in hemoglobin function during the evolution of Andean hummingbirds

Projecto-Garcia

Natarajan

Moriyama

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Animals that sustain high levels of aerobic activity under hypoxic conditions (e.g., birds that fly at high altitude) face the physiological challenge of jointly optimizing blood-O 2 affinity for O 2 loading in the pulmonary circulation and O 2 unloading in the systemic circulation. At high altitude, this challenge is especially acute for small endotherms like hummingbirds that have exceedingly high mass-specific metabolic rates. Here we report an experimental analysis of hemoglobin (Hb) function in South American hummingbirds that revealed a positive correlation between Hb-O 2 affinity and native elevation. Protein engineering experiments and ancestral-state reconstructions revealed that this correlation is attributable to derived increases in Hb-O 2 affinity in highland lineages, as well as derived reductions in Hb-O 2 affinity in lowland lineages. Site-directed mutagenesis experiments demonstrated that repeated evolutionary transitions in biochemical phenotype are mainly attributable to repeated amino acid replacements at two epistatically interacting sites that alter the allosteric regulation of Hb-O 2 affinity. These results demonstrate that repeated changes in biochemical phenotype involve parallelism at the molecular level, and that mutations with indirect, second-order effects on Hb allostery play key roles in biochemical adaptation.high-altitude adaptation | hypoxia | parallel evolution | protein evolution | epistasis

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“…Nevertheless, by changing expression conditions most globins can be expressed and purified as soluble proteins [25].…”

Section: Recombinant Expression In Escherichia Colimentioning

confidence: 99%

Exploring three different expression systems for recombinant expression of globins: Escherichia coli, Pichia pastoris and Spodoptera frugiperda

Bracke

Hoogewijs

Dewilde

2018

Analytical Biochemistry

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A B S T R A C TGlobins are among the best investigated proteins in biological and medical sciences and represent a prime tool for the study of the evolution of genes and the structure-function relationship of proteins. Here, we explore the recombinant expression of globins in three different expression systems: Escherichia coli, Pichia pastoris and the baculovirus infected Spodoptera frugiperda. We expressed two different human globin types in these three expression systems: I) the well-characterized neuroglobin and II) the uncharacterized, circular permutated globin domain of the large chimeric globin androglobin. It is clear from the literature that E.coli is the most used expression system for expression and purification of recombinant globins. However, the major disadvantage of E. coli is the formation of insoluble aggregates. We experienced that, for more complex multi-domain globins, like the chimeric globin androglobin, it is recommended to switch to a higher eukaryotic expression system.

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Expression and Purification of Recombinant Hemoglobin in Escherichia coli

Cited by 51 publications

References 40 publications

Altitudinal Variation at Duplicated β-Globin Genes in Deer Mice: Effects of Selection, Recombination, and Gene Conversion

Altitudinal Variation at Duplicated β-Globin Genes in Deer Mice: Effects of Selection, Recombination, and Gene Conversion

Repeated elevational transitions in hemoglobin function during the evolution of Andean hummingbirds

Exploring three different expression systems for recombinant expression of globins: Escherichia coli, Pichia pastoris and Spodoptera frugiperda

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