Synthesis of Recombinant Human Hemoglobin With NH2-Terminal Acetylation in Escherichia coli

Abstract

The development of new technologies for the efficient expression of recombinant hemoglobin (rHb) is of interest for experimental studies of protein biochemistry and the development of cell-free blood substitutes in transfusion medicine. Expression of rHb in Escherichia coli host cells has numerous advantages, but one disadvantage of using prokaryotic systems to express eukaryotic proteins is that they are incapable of performing post-translational modifications such as NH₂-terminal acetylation. One possible solution is to coexpress additional enzymes that can perform the necessary modifications in the host cells. Here, we report a new method for synthesizing human rHb with proper NH₂-terminal acetylation. Mass spectrometry experiments involving native and recombinant human Hb confirmed the efficacy of the new technique in producing correctly acetylated globin chains. Finally, functional experiments provided insights into the effects of NH₂-terminal acetylation on O₂ binding properties. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Gene synthesis and cloning the cassette to the expression plasmid

Basic Protocol 2: Selection of E. coli expression strains for coexpression

Basic Protocol 3: Large-scale recombinant hemoglobin expression and purification

Support Protocol 1: Measuring O₂ equilibration curves

Support Protocol 2: Mass spectrometry to confirm NH₂-terminal acetylation