Production of unmodified human adult hemoglobin in Escherichia coli.

Abstract

We have constructed a plasmid (pHE2) in which the synthetic human a-and 3-globin genes and the methionine aminopeptidase (Met-AP) gene from Escherichia coli are coexpressed under the control of separate tac promoters. The Hbs were expressed in E. coli JM109 and purified by fast protein liquid chromatography, producing two major components, a and b. Electrospray mass spectrometry shows that at least 98% and about 90% of the expressed a and (3 chains of component a, respectively, have the expected masses.The remaining 10% of the P chain in component a corresponds in mass to the .8 chain plus methionine. In component b, both a and (3 chains have the correct masses without detectable N-terminal methionine (<2%). These results have been confirmed by Edman degradation studies of the amino-terminal sequences of the a and ,B chains of these two recombinant Hb (rHb) samples. rHbs from components a and b exhibit visible optical spectra identical to that of human normal adult Hb (Hb A). Component a and Hb A have very similar oxygen-binding properties, but component b shows somewhat altered oxygen binding, especially at low pH values. 1H-NMR spectra of component a and Hb A are essentially identical, whereas those of component b exhibit altered ring current-shifted and hyperfine-shifted proton resonances, indicating altered heme conformation in the (3 chain. These altered resonance patterns can be changed to those of Hb A by converting component b to the ferric state and then to the deoxy state and finally back to either the carbonmonoxy or oxy form. Thus, our E. coli expression system produces native, unmodified Hb A in high yield and can be used to produce desired mutant Hbs.To make use of our ability to rationally design mutant human Hbs needed for research on structure-function relationships, an efficient expression system for producing unmodified human Hbs in high yields is needed. Human adult Hb (Hb A) is a tetrameric protein containing two a chains and two (3 chains having 141 and 146 amino acid residues each, respectively. Human globins and Hbs have been expressed in transgenic mice (1-4), transgenic swine (5), insect cell cultures (6), yeast (7, 8), and Escherichia coli (9-11). In many respects, the E. coli system is the best choice for our purposes because of its high expression efficiency and the ease ofperforming site-directed mutagenesis. The first E. coli system to express human a-and 3-globin as a fusion protein was developed by Nagai and Th0gersen (9,12), but the product processing procedure is very laborious and gives low yield. Thus, this expression system has limitations, especially when large amounts of recombinant Hb (rHb) are required for biochemical-biophysical studies. Hoffman et al.

Sickle Cell Hemoglobin with Mutation at αHis-50 Has Improved Solubility

“…Cells were grown in DM-4 medium (25) at 30°C in a 20-liter fermenter (B. Braun Biotech International, model Biostat C). The induction and purification of rHb were carried out as described previously (26). Hb A and Hb S from whole blood were purified according to established methods in our laboratory (27,28).…”

Role of β112 Cys (G14) in Homo- (β4) and Hetero- (α2β2) Tetramer Hemoglobin Formation

“…Plasmids were transfected into E. coli (JM 109) (Promega Co.), bacteria were grown at 30°C, and soluble ␤-globin chain variants were isolated as described (5,8). Expression and purification of ␤-globin variants were basically as described previously (5,8). However, cationexchange chromatography on a Source 15 S column (Amersham Pharmacia Biotech) instead of Superose 12 gel filtration was used for purification of the ␤ chain variants.…”

“…␤112 Ser, ␤112 Val, ␤112 Thr, and ␤112 Asp) were constructed and expressed using the pHE2␤ plasmid vector that contains cDNAs coding for each ␤ chain variant and methionine aminopeptidase which was originally developed to express ␣ and ␤ chains at the same time (8,9). The basic strategy for generation of these variants by site-specific mutagenesis of the normal ␤ chain involves recombination/polymerase chain reaction as described previously (10).…”

“…Clones were subjected to DNA sequence analysis of the entire ␤-globin cDNA region using site-specific primers and fluorescently tagged terminators in a cycle sequencing reaction in which extension products were analyzed on an automated DNA sequencer. Plasmids were transfected into E. coli (JM 109) (Promega Co.), bacteria were grown at 30°C, and soluble ␤-globin chain variants were isolated as described (5,8). Expression and purification of ␤-globin variants were basically as described previously (5,8).…”

Assembly of functional skeletal muscle troponin complex in Escherichia coli

“…It has been reported that the assembly of myosin heavy and light chains occurs in E. coli, but the protein has not been fully characterized (McNally et al, 1988). The two subunits of hemoglobin have been coexpressed in E. coli and shown to renature into a functional tetrameric complex (Hoffman et al, 1990;Shen et al, 1993). The overexpression of the chaperones in E. coli cells expressing human procollagenase results in an increase in the accumulation and solubility of this protein in the cytoplasm, but it is not clear if the resulting protein is functional (Lee and Olins, 1992).…”