X-ray crystallographic and functional studies of human haemoglobin mutants produced in Escherichia coli.
Human beta-globin was produced in Escherichia coli as a cleavable fusion protein using the expression vector pLcII [Nagai and Thøgersen, Nature 301, p. 810, 1984]. The fusion protein CIIFX beta-globin was purified under denaturing conditions to homogeneity and the authentic beta-globin was liberated by blood coagulation factor Xa. beta-Globin was then folded and reconstituted with haem and alpha-subunits to form fully functional alpha 2 beta 2-tetramers [Nagai et al., Proc. natn. Acad. Sci. USA 82, p. 7252, 1985]. This has enabled us to produce mutants with amino acid substitutions in the beta-subunit at will and in sufficient quantities to study their oxygen-binding properties and three-dimensional structures. We have crystallised three mutants, Hb Nympheas [Cys-93 beta----Ser], Hb Daphne [Cys-93 beta----Ser, His-143 beta----Arg] and Hb Sandra [Cys-93 beta----Ser, Asp-94 beta----Glu], and have solved their structures to high resolution by x-ray crystallography.
D000595:Amino Acid Sequence; D000818:Animals; D005075:Biological Evolution; D004926:Escherichia coli / Q000378:metabolism*; D005399:Fishes / Q000097:blood; D005821:Genetic Techniques; D006454:Hemoglobins / Q000235:genetics* / Q000502:physiology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009154:Mutation; D010108:Oxyhemoglobins / Q000378:metabolism; D014961:X-Ray Diffraction
D006454:Hemoglobins; D010108:Oxyhemoglobins