Figure 1.
Design of the expression plasmids.
As shown in Panel A, the pGM-HαHβ plasmid system contains a cassette of tandemly arrayed α- and β-globin genes with a Shine-Dalgarno ribosomal binding site as a spacer DNA. The MAP cassette is located downstream of β-globin. As shown in panel B, the pCO-MAP plasmid contains a MAP cassette under the control of a T7 promoter along with a kanamycin resistance gene.
Figure 2.
SDS-PAGE (20%) image showing the purified rHb isoforms.
Lane BHb, Bovine Hb standard; Lane M, BioRad size standards; Lane 1, HH crude lysate; Lane 2, HH clarified fraction after dialysis; Lane 3, HH purified fraction; Lane 4, LL crude lysate; Lane 5, LL clarified fraction after dialysis; Lane 6, LL purified fraction; Lane NaHb, Native Hb from deer mouse blood.
Figure 3.
Variation in the efficiency of MAP enzyme in cleaving N-terminal methionine residues from the α- and β-chain subunits of purified HH rHb.
The relative fractions of methionine and valine in the N-terminal residue positions are shown for three experimental conditions: (A) BL21Star™ (DE3) at 12°C for 24 hr, (B) BL21Star™ (DE3) at 30°C for 4 hr with co-expression of pCO-MAP expression plasmid, and (C) JM109 (DE3) at 28°C for 16 hr with co-expression of pCO-MAP expression plasmid. The efficiency of the MAP enzyme in cleaving the N-terminal methione residues from the α- and β-chain subunits is indicated by the relative fraction of methionine vs. valine in the N-terminal residue position. When the percentage of globin chains with NA1 valine exceeds 95%, this indicates that the post-translational modification has been carried out properly.
Figure 4.
O2-binding properties of purified deer mouse rHbs.
Panels A and B show O2 equilibrium curves for purified rHbs HH and LL, respectively. The rHbs HH and LL were expressed in the JM109 (DE3) E. coli strain at 28°C for 16 hr with co-expression of the pCO-MAP plasmid. O2-binding measurements were conducted at pH 7.4 at 37°C in presence and absence of allosteric cofactors (0.1 M Cl−, 0.1 M Hepes, 2.0-fold ratio DPG /rHb tetramers and 0.2 mM heme). Horizontal lines denote the half-saturation value for each rHb.