Expression of Soluble Human β-Globin Chains in Bacteria and Assembly in Vitro with α-Globin Chains

Abstract

Authentic soluble human ␤-globin chains were produced in Escherichia coli using an expression plasmid (pHE2␤) containing full-length cDNAs coding for human ␤-globin chain and methionine aminopeptidase. Spectral properties of the purified ␤-globin were identical to those of authentic ␤-globin. Soluble ␤-globin showed low (16 kDa) and high molecular mass (32 kDa) forms that could be separated by gel filtration chromatography. SDS-polyacrylamide gel electrophoresis and electrospray mass spectrometry revealed the 32-kDa species was dimeric ␤-globin formed by an intermolecular disulfide bond, while the 16-kDa species was authentic monomeric ␤-globin. Monomeric forms of ␤-globin, like authentic native ␤-globin, formed tetrameric hemoglobin (Hb) A (␣ 2 ␤ 2 ) in vitro upon incubation with ␣-globin, while dimeric forms did not. When ␤-globin dimers, however, were converted to monomers by incubation with dithiothreitol, the ␤-globin chain monomers assembled with ␣-globin and formed hemoglobin tetramers. ␣-Globin was more thermally unstable than ␤-globin, while assembled tetramers promoted higher stability. Disulfide-bonded ␤-globin dimers showed a slight increase in thermal stability compared with ␤-globin; however, dimers were still more unstable than tetrameric Hb A. These results indicate that presence of ␣ chains favors assembly with ␤-globin, ␤-␤ dimers cannot bind ␣ chains, and that Hb A tetramer formation results in the most thermally stable species.The development of molecular biological techniques to replace selectively individual amino acids has helped further our understanding of the relationship between structure and function of hemoglobin. Initial reports described production of normal and modified human ␤-globin in bacteria employing a fusion protein expression vector (1). An expression system was later developed in which ␣-and ␤-globin chains were coexpressed, resulting in formation of soluble tetrameric hemoglobin in yeast (2, 3). Recent studies described coexpression of human ␣-and ␤-globin in Escherichia coli, which resulted in formation of soluble tetrameric hemoglobins (4), as well as a system for high expression of insoluble ␤-globin chains in E. coli. (5). These last two systems, however, result in globin chains containing N-terminal methionine, which may affect functional properties of hemoglobin. More recently, Shen et al. (6) expressed soluble hemoglobin tetramers lacking N-terminal methionine in bacteria by coexpression of ␣-and ␤-globin cDNAs with methionine aminopeptidase cDNA.In order to further the understanding of hemoglobin assembly and folding, production of soluble single-chain hemoglobin variants is critical; however, expression of recombinant, soluble, individual globin chains has not been realized to date (4,5,7,8). Globins expressed in cells with and without additional hemin often form insoluble inclusion bodies that require harsh denaturing conditions for solubilization. After solubilization, chains must then be renatured in vitro and form correctly folded native globin chains, which...

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

“…Further purification was achieved using cation-exchange chromatography on a Source 15 S column. Purified ␤ A chains migrate predominantly as 32-kDa dimers (9), whereas the four ␤112 variants migrate mainly as 16-kDa monomers with small traces of dimers on SDS-PAGE (Fig. 1).…”

“…In addition, Ser and Thr are hydrophilic like Cys, whereas Val is highly hydrophobic. After DNA sequence confirmation, the three ␤-globin chain variants were expressed in bacteria and purified by a combination of two anion-exchange chromatography steps using DEAE-cellulose and Mono-Q columns (9). Further purification was achieved using cation-exchange chromatography on a Source 15 S column.…”

“…␤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).…”

Folding and assembly of hemoglobin monitored by electrospray mass spectrometry using an on-line dialysis system

“…Such a process would have to involve (1) the folding of individual subunits, (2) subunit binding to heme, and (3) subunit assembly to form tetrameric Hb (obviously, the three steps do not necessarily have to occur in this particular order). Previous attempts to carry out related experiments were frustrated by very low heterotetramers yields due to extensive aggregation and precipitation [55]. In fact, aggregation is a common problem even for reconstitution schemes employing folded ␣-and ␤-subunits [45].°This°could°imply that Hb folding and self-assembly necessarily requires a chaperone system, and possibly other components of the cellular machinery.…”

Assembly of γ- with α-Globin Chains to Form Human Fetal Hemoglobin in Vitro and in Vivo

“…authentic human Hb A lacking the chain-initiating N-terminal methionines (11). Using this system we previously expressed individual ␤-globin chains after removing the ␣-globin cDNA fragment (12). To achieve expression of individual G␥-globin chains and Hb F, the human ␥-globin cDNA (441 bp) was obtained by PCR from pGS 389␥, which we previously used as a yeast expression vector and which contains ␣-and ␥-globin cDNAs (13).…”