Production of human normal adult and fetal hemoglobins in Escherichia coli

Abstract

A hemoglobin expression system in Escherichia coli is described. In order to produce authentic human hemoglobin, we need to co-express both methionine aminopeptidase and globin genes under the control of a strong promoter. We have constructed three plasmids, pHE2, pHE4 and pHE7, for the expression of human normal adult hemoglobin and a plasmid, pHE9, for the expression of human fetal hemoglobin, in high yields. The globin genes can be derived from either synthetic genes or human globin cDNAs. The extra amino-terminal methionine residues of the expressed globins can be removed by the co-expressed methionine aminopeptidase. The heme is inserted correctly into the expressed alpha-globin from our expression plasmids. A fraction (approximately 25%) of the heme is not inserted correctly into the expressed beta- or gamma-globin. However, the incorrectly inserted hemes can be converted into the correct conformation by carrying out a simple oxidation-reduction process on the purified hemoglobin molecule. We have investigated the functional properties of the expressed hemoglobins by measuring their oxygen-binding properties and their structural features by obtaining their 1H-NMR spectra. Our results show that authentic human normal adult and fetal hemoglobins can be produced from our expression plasmids in E. coli and in high yields. Our expression system allows us to design and to produce any recombinant hemoglobins needed for our research on the structure-function relationship in hemoglobin.

Distal Histidine Stabilizes Bound O2 and Acts as a Gate for Ligand Entry in Both Subunits of Adult Human Hemoglobin

“…This wild-type recombinant Hb control has R state rate parameters that are virtually identical to those of native R state HbA (28). The Leu(E7) mutants were also expressed from the pHE2 plasmid designed by Ho and co-workers (29,30) to examine the effects of the V1M mutation (see supplemental Table S1). Individual ␣ and ␤ mutants were separated and isolated using procedures initially developed by Parkhurst and Parkhurst (31) (see supplemental Section A).…”

Development of Recombinant Hemoglobin-Based Oxygen Carriers

“…A few years later, T-J. Shen and C. Ho developed a system in which a-and bchains containing an extra N-terminal Met were coexpressed with E. coli methionine aminopeptidase to allow complete removal of the initiator amino acid and generation of recombinant Hb identical in a primary structure to native HbA (136,137). The Somatogen and Nagai groups also developed a fused di-a gene, which was inserted in an operon with a copy of the b-gene to express a tetramer that does not dissociate into a 1 b 1 dimers under normal physiological conditions, even when very dilute (79).…”

“…Chien Ho's group for producing recombinant Hb in E. coli (137). Stimulation of endogenous heme synthesis by E. coli can be achieved by the addition of d-aminolevulonic acid, but this reagent is too expensive to use routinely for large-scale production, and the enhancement of rHb production is not as great as adding high levels of exogenous hemin.…”

The Proteomics of N-terminal Methionine Cleavage

“…To this aim, we therefore added an option relative to the overexpression of any favorite protein in the TermiNator2 prediction tool. If the protein has an N terminus associated with a risk of nonprocessing, various methods may be used to prevent Met retention: (i) adjusting expression conditions to optimize MAP activity according to the rate of protein synthesis in vivo (58), (ii) in vitro processing with purified wild-type or engineered MAP (59 -62), (iii) dual co-overproduction of MAP and the protein of interest in vivo (25,63,64), (iv) processing in vitro or in vivo of the N terminus with another amino-or endoprotease (65-67), or (v) fusion of the ORF to a propeptide or ubiquitin sequence and use of endogenous signal peptidase specificity (68,69). This last strategy has also proved successful for residues that cannot be unmasked by NME (this study, Fig.…”