Expression of soluble human β-globin chains in bacteria and assembly in vitro with α-globin chains

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Abstract

Authentic soluble human β-globin chains were produced in Escherichia coli using an expression plasmid (pHE2β) containing full-length cDNAs coding for human β-globin chain and methionine aminopeptidase. Spectral properties of the purified β-globin were identical to those of authentic β-globin. Soluble β-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 β-globin formed by an intermolecular disulfide bond, while the 16-kDa species was authentic monomeric β-globin. Monomeric forms of β-globin, like authentic native β-globin, formed tetrameric hemoglobin (Hb) A (α2β2) in vitro upon incubation with α-globin, while dimeric forms did not. When β-globin dimers, however, were converted to monomers by incubation with dithiothreitol, the β-globin chain monomers assembled with α-globin and formed hemoglobin tetramers. α-Globin was more thermally unstable than β-globin, while assembled tetramers promoted higher stability. Disulfide-bonded β-globin dimers showed a slight increase in thermal stability compared with β-globin; however, dimers were still more unstable than tetrameric Hb A. These results indicate that presence of a chains favors assembly with β-globin, β-β dimers cannot bind a chains, and that Hb A tetramer formation results in the most thermally stable species.

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Yamaguchi, T., Pang, J., Reddy, K. S., Witkowska, H. E., Surrey, S., & Adachi, K. (1996). Expression of soluble human β-globin chains in bacteria and assembly in vitro with α-globin chains. Journal of Biological Chemistry, 271(43), 26677–26683. https://doi.org/10.1074/jbc.271.43.26677

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