Expression, identification and purification of human FMRP isoform 10.
Fragile X syndrome (FXS), which is the most frequently inherited mental retardation after Down syndrome, is caused by the absence of the fragile X mental retardation protein (FMRP) encoded by the fragile X mental retardation 1 (FMR1) gene. Patients with FXS can be identified by antibody tests that detect the absence of FMRP caused by loss-of-function mutations including the prevalent CGG repeat amplification in lymphocytes. Although the expression of recombinant FMRP in prokaryotic and eukaryotic expression systems has been achieved in different laboratories, the solubilization and purification of this protein is time consuming, varies with each protocol, and often results in low yield. In this study, glutathione S-transferase FMRP fusion protein (GST-FMRP) was expressed in and purified from Escherichia coli BL21(DE3) pLysS cells transformed with pGEX-6P-1 fusion expression vector containing the FMR1 cDNA. The recombinant GST-FMRP was purified on a glutathione sepharose 4B affinity column and detected using SDS-PAGE followed by western blotting with anti-FMRP antibody. This highly purified and soluble GST-FMRP protein can be very beneficial for generating anti-FMRP antibodies and developing FXS diagnosis kits.
Department of endocrinology, National Research Institute for Family Planning, Beijing, 100081, China.
Yong
Y
Guo
Fangfang
F
Li
Fangfang
F
Cui
Ning
N
Ding
Rong
R
Zhang
Lin-na
LN
Liu
Jianping
J
Zou
D001483:Base Sequence; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D051860:Fragile X Messenger Ribonucleoprotein 1 / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D016133:Polymerase Chain Reaction; D020033:Protein Isoforms / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D017931:DNA Primers; D051860:Fragile X Messenger Ribonucleoprotein 1; D020033:Protein Isoforms; D011994:Recombinant Proteins; C497683:FMR1 protein, human