Adachi K, Yamaguchi T, Yang Y, Konitzer P T, Pang J, Reddy K S, Ivanova M, Ferrone F, Surrey S
Division of Hematology, Children's Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
Protein Expr Purif. 2000 Oct;20(1):37-44. doi: 10.1006/prep.2000.1277.
Individual, soluble human alpha-globin chains were expressed in bacteria with exogenous heme and methionine aminopeptidase. The yields of soluble alpha chains in bacteria were comparable to those of recombinant non-alpha chains expressed under the same conditions. Molecular mass and gel-filtration properties of purified recombinant alpha chains were the same as those of authentic human alpha chains. Biochemical and biophysical properties of isolated alpha chains were identical to those of native human alpha chains as assessed by UV/vis, circular dichroism (CD), and nuclear magnetic resonance (NMR) spectroscopy which contrasts with previous results of refolded precipitated alpha chains made in the presence of heme in vitro (M. T. Sanna et al., J. Biol. Chem. 272, 3478-3486, 1997). Mixtures of purified, soluble recombinant alpha-globin and native beta-globin chains formed heterotetramers in vitro, and oxygen- and CO-binding properties as well as the heme environment of the assembled tetramers were experimentally indistinguishable from those of native human Hb A. UV/vis, CD, and NMR spectra of assembled Hb A were also the same as those of human Hb A. These results indicate that individual expressed alpha chains are stable in bacteria and fold properly in vivo and that they then can assemble with free beta chains to form hemoglobin heterotetramers in vivo as well as in vitro.
单个可溶性人α-珠蛋白链在含有外源性血红素和甲硫氨酸氨肽酶的细菌中表达。细菌中可溶性α链的产量与在相同条件下表达的重组非α链相当。纯化的重组α链的分子量和凝胶过滤特性与天然人α链相同。通过紫外/可见光谱、圆二色性（CD）和核磁共振（NMR）光谱评估，分离的α链的生化和生物物理特性与天然人α链相同，这与之前在体外血红素存在下重折叠沉淀的α链的结果形成对比（M. T. Sanna等人，《生物化学杂志》272，3478 - 3486，1997）。纯化的可溶性重组α-珠蛋白和天然β-珠蛋白链的混合物在体外形成异源四聚体，组装后的四聚体的氧结合和一氧化碳结合特性以及血红素环境在实验上与天然人血红蛋白A无法区分。组装后的血红蛋白A的紫外/可见光谱、CD光谱和NMR光谱也与人类血红蛋白A相同。这些结果表明，单个表达的α链在细菌中是稳定的，在体内能正确折叠，然后它们可以与游离的β链组装形成血红蛋白异源四聚体，无论是在体内还是体外。
