Overexpression, refolding, purification of Erbin PDZ domain from Escherichia coli and preparation of its polyclonal antibody.
The Erbin was recently identified. The antibody against Erbin has not been commercially available. As a new member of peripheral protein LAP family and novel type of adaptor protein, its functions and binding partners are not completely known. In the present study, cDNA encoding PDZ domain of Erbin was inserted in a prokaryotic expression vector. His-tagged recombinant protein was overproduced in E. coli and purified by Ni-NTA column chromatography. About 14.4 mg of the purified protein was obtained from 500 mL of cell culture. The purity of the recombinant protein was higher than 90%. The polyclonal antibody against this protein was raised. The antibody can recognize both denatured and natural Erbin protein. It will be used to further identify the new binding partners of Erbin and study its unknown functions.
Zhang
H
H
Institute of Basic Medical Sciences, Beijing, P.R. China.
Yu
M
M
Hu
M
M
Qian
L
L
Chen
L
L
Shi
M
M
Shen
B
B
Guo
N
N
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D054731:PDZ Domains; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015854:Up-Regulation
D048868:Adaptor Proteins, Signal Transducing; D000911:Antibodies, Monoclonal; C413364:ERBIN protein, human; D011994:Recombinant Proteins