title: Expression and purification of recombinant hemoglobin in Escherichia coli.
abstract: BACKGROUND
  Recombinant DNA technologies have played a pivotal role in the elucidation of structure-function relationships in hemoglobin (Hb) and other globin proteins. Here we describe the development of a plasmid expression system to synthesize recombinant Hbs in Escherichia coli, and we describe a protocol for expressing Hbs with low intrinsic solubilities. Since the α- and β-chain Hbs of different species span a broad range of solubilities, experimental protocols that have been optimized for expressing recombinant human HbA may often prove unsuitable for the recombinant expression of wildtype and mutant Hbs of other species.
  
  
  RESULTS
  As a test case for our expression system, we produced recombinant Hbs of the deer mouse (Peromyscus maniculatus), a species that has been the subject of research on mechanisms of Hb adaptation to hypoxia. By experimentally assessing the combined effects of induction temperature, induction time and E. coli expression strain on the solubility of recombinant deer mouse Hbs, we identified combinations of expression conditions that greatly enhanced the yield of recombinant protein and which also increased the efficiency of post-translational modifications.
  
  
  CONCLUSIONS
  Our protocol should prove useful for the experimental study of recombinant Hbs in many non-human animals. One of the chief advantages of our protocol is that we can express soluble recombinant Hb without co-expressing molecular chaperones, and without the need for additional reconstitution or heme-incorporation steps. Moreover, our plasmid construct contains a combination of unique restriction sites that allows us to produce recombinant Hbs with different α- and β-chain subunit combinations by means of cassette mutagenesis.
authors.affiliation: School of Biological Sciences, University of Nebraska, Lincoln, Nebraska, United States of America.
authors.forename: Chandrasekhar
authors.initials: C
authors.lastname: Natarajan
authors.forename: Xiaoben
authors.initials: X
authors.lastname: Jiang
authors.forename: Angela
authors.initials: A
authors.lastname: Fago
authors.forename: Roy E
authors.initials: RE
authors.lastname: Weber
authors.forename: Hideaki
authors.initials: H
authors.lastname: Moriyama
authors.forename: Jay F
authors.initials: JF
authors.lastname: Storz
mesh_terms: D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006454:Hemoglobins / Q000235:genetics* / Q000302:isolation & purification; D016296:Mutagenesis; D010542:Peromyscus; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification
chemical_list: D006454:Hemoglobins; D011994:Recombinant Proteins