High-level production of recombinant Arenicola marina globin chains in Escherichia coli: a new generation of blood substitute.
This work reports for the first time the expression of a soluble B2 globin chain that is part of the extracellular hexagonal-bilayer haemoglobin from Arenicola marina. Two recombinant B2 globins were produced, one fused with gluthatione S-tranferase (B2-GST) and the other without a fusion tag (RecB2) and requiring a different purification procedure. We also describe a new method for the expression of globin that uses Studier's auto-induction medium together with the heme precursor delta-aminolevulinic acid. Media supplementation with the heme precursor delta-aminolevulinic acid in the culture increased heme synthesis by E. coli leading to the expression of the recombinant B2 globins in their active form. RecB2 and B2-GST were expressed with a yield of up to 105 mg/l of E. coli culture. Our approach is rapid and requires only one chromatographic purification step for B2-GST and three purification steps for RecB2. The overall results on RecB2 and B2-GST show that the recombinant globins exhibit similar properties to those of Arenicola marina native HBL-Hb with a great stability and a strong oxygen binding. The results and methodologies described in this paper are the beginning of a work aiming at reconstituting a recombinant HBL-Hb by genetic engineering in order to produce an innovative oxygen carrier for therapeutic applications.
Harnois
Thomas
T
UPMC Univ Paris 06, Equipe Ecophysiologie, Adaptation et Evolution Moleculaires, Roscoff, France.
Rousselot
Morgane
M
Rogniaux
Hélène
H
Zal
Franck
F
D000328:Adult; D000595:Amino Acid Sequence; D000622:Aminolevulinic Acid / Q000378:metabolism; D000818:Animals; D001802:Blood Substitutes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D005914:Globins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D006418:Heme / Q000378:metabolism; D006454:Hemoglobins / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011077:Polychaeta; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D013056:Spectrophotometry, Ultraviolet
D001802:Blood Substitutes; D006454:Hemoglobins; D011993:Recombinant Fusion Proteins; D006418:Heme; D000622:Aminolevulinic Acid; D005914:Globins