The 22.5 kDa spectrin-binding domain of ankyrinR binds spectrin with high affinity and changes the spectrin distribution in cells in vivo.
It was previously shown that ankyrins play a crucial role in the membrane skeleton arrangement. Purifying ankyrinR obtained from erythrocytes is a time-consuming process. Therefore, cloned and bacterially expressed ankyrinR-spectrin-binding domain (AnkSBD) is a demanded tool for studying spectrin-ankyrin interactions. In this communication, we report on the cloning and purification of AnkSBD and describe the results of binding experiments, in which we showed high-affinity interactions between the AnkSBD construct and isolated erythrocyte or non-erythroid spectrins. pEGFP-AnkSBD-transfected cells co-localised with non-erythroid spectrin in HeLa cells. The functional interactions of the AnkSBD construct in vivo and in vitro open many possibilities to study the structure and function of this domain, which has not yet been as extensively studied when compared to the aminoterminal domain of this protein.
Kolondra
Adam
A
Laboratory of Cytobiochemistry, Biotechnology Faculty, University of Wroclaw, ul Przybyszewskiego 63/77, 51148 Wroclaw, Poland.
Grzybek
Michal
M
Chorzalska
Anna
A
Sikorski
Aleksander F
AF
D017487:Ankyrins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D013049:Spectrin / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D017487:Ankyrins; D017931:DNA Primers; D018076:DNA, Complementary; D013049:Spectrin