Expression and purification of recombinant cytoplasmic domain of human erythrocyte band 3 with hexahistidine tag or chitin-binding tag in Escherichia coli.
The cytoplasmic domain of erythrocyte band 3 (cdb3) serves as a center of membrane organization in the erythrocytes by its interaction with multiple proteins including ankyrin, protein 4.1, protein 4.2, hemoglobin, and several glycolytic enzymes. In this paper, human cdb3 was cloned into three different expression vectors controlled by T7 polymerase promoter and induced with isopropyl beta-D-thiogalactopyranoside in Escherichia coli. Two of the fusion proteins containing hexahistidine tag in the N-terminal or C-terminal were purified by immobilized metal affinity column chromatography. The third recombinant cdb3 containing the affinity chitin-binding tag was purified using chitin beads followed by specific self-cleavage, which released cdb3 according to the mechanism of protein splicing. The molecular weights of purified recombinant proteins were verified by mass spectrometry. The pH-dependent properties of the intrinsic tryptophan fluorescence of the three kinds of recombinant cdb3 were compared with that of the cdb3 extracted from the erythrocytes, showing that there were no significant differences between them. Using pull-down assay combined with Western blot analysis, the interaction between recombinant cdb3 and protein 4.2 C3 fragment was verified. These demonstrated that the recombinant proteins were both structurally and functionally active. The typical yields of cdb3 purified with hexahistidine tag in the N-terminal, C-terminal, and cleaved from chitin bead were 10.6, 9.6, and 1.5 mg from 1L culture medium, respectively.
Ding
Yu
Y
Department of Physiology and Biophysics, School of Life Sciences, Fudan University, Shanghai 200433, China.
Jiang
Weihua
W
Su
Yang
Y
Zhou
Hanqing
H
Zhang
Zhihong
Z
D002686:Chitin / Q000737:chemistry; D004910:Erythrocyte Membrane / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine / Q000737:chemistry; D006801:Humans; D013058:Mass Spectrometry; D008562:Membrane Glycoproteins / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011971:Receptors, Immunologic / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D063267:Sialic Acid Binding Ig-like Lectin 1; D014364:Tryptophan / Q000032:analysis
D008562:Membrane Glycoproteins; D011971:Receptors, Immunologic; D011993:Recombinant Fusion Proteins; C571166:SIGLEC1 protein, human; D063267:Sialic Acid Binding Ig-like Lectin 1; D002686:Chitin; D006639:Histidine; D014364:Tryptophan