Expression, purification, and characterization of the functional dimeric cytoplasmic domain of human erythrocyte band 3 in Escherichia coli.
The cytoplasmic domain of the human erythrocyte membrane protein, band 3 (cdb3), contains binding sites for hemoglobin, several glycolytic enzymes, band 4.1, band 4.2, and ankyrin, and constitutes the major linkage between the membrane skeleton and the membrane. Although erythrocyte cdb3 has been partially purified from proteolyzed red blood cells, further separation of the water-soluble 43-kDa and 41-kDa proteolytic fragments has never been achieved. In order to obtain pure cdb3 for crystallization and site-directed mutagenesis studies, we constructed an expression plasmid that has a tandemly linked T7 promoter placed upstream of the N-terminal 379 amino acids of the erythrocyte band 3 gene. Comparison of several Escherichia coli strains led to the selection of the BL21 (DE3) strain containing the pLysS plasmid as the best host for efficient production of cdb3. About 10 mg of recombinant cdb3 can be easily purified from 4 L of E. coli culture in two simple steps. Comparison of cdb3 released from the red blood cell by proteolysis with recombinant cdb3 reveals that both have the same N-terminal sequence, secondary structure, and pH-dependent conformational change. The purified recombinant cdb3 is also a soluble stable dimer with the same Stokes radius as erythrocyte cdb3. The affinities of the two forms of cdb3 for ankyrin are essentially identical; however, recombinant cdb3 with its unblocked N-terminus exhibits a slightly lower affinity for aldolase.
Wang
C C
CC
Department of Chemistry, Purdue University, West Lafayette, Indiana 47907.
Badylak
J A
JA
Lux
S E
SE
Moriyama
R
R
Dixon
J E
JE
Low
P S
PS
D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001667:Binding, Competitive; D003001:Cloning, Molecular; D004910:Erythrocyte Membrane / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping
D001457:Anion Exchange Protein 1, Erythrocyte; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins