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Expression, purification, and characterization of the functional dimeric cytoplasmic domain of human erythrocyte band 3 in Escherichia coli by Cheng C. Wang; Ryuichi Moriyama; Philip S. Low; Janine A. Badylak; Jack E. Dixon; Samuel E. Lux is a Biochemistry, Genetics and Molecular Biology article available to read on EtoBox.
What is Expression, purification, and characterization of the functional dimeric cytoplasmic domain of human erythrocyte band 3 in Escherichia coli about?
## Abstract 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
Who reads Expression, purification, and characterization of the functional dimeric cytoplasmic domain of human erythrocyte band 3 in Escherichia coli?
It is typically read by researchers, students, and practitioners in Biochemistry, Genetics and Molecular Biology.
- Author
- Cheng C. Wang; Ryuichi Moriyama; Philip S. Low; Janine A. Badylak; Jack E. Dixon; Samuel E. Lux
- Publisher
- Cold Spring Harbor Laboratory Press; Wiley (John Wiley & Sons); Wiley-Blackwell; Wiley (ISSN 0961-8368)
- Published
- 1992
- Language
- EN
- Field
- Biochemistry, Genetics and Molecular Biology (Life Sciences)