Expression and subcellular localization of P9-ZFD protein in patients with myasthenia gravis.
OBJECTIVE
To express and purify the protein coded by the TRAF-type zinc finger domain of myasthenia gravis (MG)-related gene P9 (P9-ZFD) and to prepare P9-ZFD antiserum for detecting expression and subcellular distribution of P9-ZFD protein in the skeletal muscles of patient with MG.


METHODS
The cDNA encoding P9-ZFD was amplified by RT-PCR. The cloned P9-ZFD cDNA was ligated into pET24a, and the P9-ZFD recombinant protein was induced via E. coli. BL21 (DE3) and purified by histidine affinity chromatography. P9-ZFD antiserum was prepared and its titer and specificity were determined by ELISA and Western blot. Expression and subcellular distribution of P9-ZFD protein in the skeletal muscles of MG and control were studied.


RESULTS
The molecular weight of purified P9-ZFD protein was about 30 kD. Its purity was more than 95%. Antiserum specific for P9-ZFD was excellent. P9-ZFD protein is fully confined to the cytoplasm membrane of skeletal muscle cell of MG, obvious immunostaining was absent in the A, I, and Z bands of cytoplasm and no immunoreactivity was observed in the skeletal muscle cell of control.


CONCLUSIONS
P9-ZFD protein is expressed as a cytoplasm membrane-bound protein and has obvious distribution difference in the skeletal muscle cells of patient with MG and normal control.
Ren
Ming-shan
MS
Department of Neurology, Huashan Hospital of Fudan University, Shanghai 200040. rmingshan@msn.com
Lu
Chuan-zhen
CZ
Qiao
Jian
J
Ren
Hui-min
HM
Xu
Ren
R
Gan
Ren-bao
RB
D000328:Adult; D002462:Cell Membrane / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005260:Female; D006801:Humans; D009124:Muscle Proteins / Q000096:biosynthesis* / Q000235:genetics; D018482:Muscle, Skeletal / Q000378:metabolism* / Q000473:pathology; D009157:Myasthenia Gravis / Q000378:metabolism*; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection; D016335:Zinc Fingers
D009124:Muscle Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins