[Frontiers in Bioscience E4, 2579-2583, June 1, 2012]

Expression, identification and purification of human FMRP Isoform 10

Yong Guo1, 2, Fangfang Li3, Fangfang Cui4, Ning Ding1, 2, Rong Zhang3, Lin-na Liu5, Jianping Zou1, 2

1Department of endocrinology, National Research Institute for Family Planning, Beijing, 100081, China, 2WHO Collaborative Center for Research in Human Reproduction, Beijing, 100081, China, 3School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang, 110016, China, 4Pharmaceutical College of Henan University, Kaifeng, 475000, China, 5Department of Pharmacy, Tangdu Hospital, Fourth Military Medical University, Xi'an 710038, China

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Materials and methods
3.1. Reagents
3.2. Construction and identification of pGEX-6P-1-FMR1 recombinant plasmid
3.3. Expression of GST-FMRP in E.coli
3.4. Purification of Recombinant FMRP
4. Results and discussion
4.1. Construction and identification of pGEX-6P-1-FMR1-ISO10
4.2. Expression of GST-FMRP ISO10 in E.coli
4.3. Purification of FMRP ISO10
5. Acknowledgments
6. References

1.ABSTRACT

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.